U.S. patent application number 10/206155 was filed with the patent office on 2003-08-21 for use of glycosylceramides as adjuvants for vaccines against infections and cancer.
This patent application is currently assigned to NEW YORK UNIVERSITY. Invention is credited to Gonzalez-Aseguinolaza, Gloria, Koezuka, Yasuhiko, Tsuji, Moriya.
Application Number | 20030157135 10/206155 |
Document ID | / |
Family ID | 23192348 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030157135 |
Kind Code |
A1 |
Tsuji, Moriya ; et
al. |
August 21, 2003 |
Use of glycosylceramides as adjuvants for vaccines against
infections and cancer
Abstract
The present invention relates to methods and compositions for
augmenting an immunogenicity of an antigen in a mammal, comprising
administering said antigen together with an adjuvant composition
that includes glycosylceramide, preferably
.alpha.-galactosylceramide (.alpha.-GalCer). According to the
present invention, the use of glycosylceramide as an adjuvant is
attributed at least in part to the enhancement and/or extension of
antigen-specific Th1-type responses, in particular, CD8+ T cell
responses. The methods and compositions of the present invention
can be useful for prophylaxis and treatment of various infectious
and neoplastic diseases.
Inventors: |
Tsuji, Moriya; (New York,
NY) ; Gonzalez-Aseguinolaza, Gloria; (Navarra,
ES) ; Koezuka, Yasuhiko; (Gunma, JP) |
Correspondence
Address: |
DARBY & DARBY P.C.
P. O. BOX 5257
NEW YORK
NY
10150-5257
US
|
Assignee: |
NEW YORK UNIVERSITY
|
Family ID: |
23192348 |
Appl. No.: |
10/206155 |
Filed: |
July 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60308056 |
Jul 25, 2001 |
|
|
|
Current U.S.
Class: |
424/278.1 ;
424/204.1; 435/6.16; 435/7.1; 514/429 |
Current CPC
Class: |
A61P 31/10 20180101;
Y02A 50/412 20180101; A61P 31/12 20180101; A61K 39/39 20130101;
A61K 2039/57 20130101; A61P 35/00 20180101; A61P 31/06 20180101;
C12N 2740/16122 20130101; C07K 14/445 20130101; A61K 2039/55511
20130101; A61P 31/18 20180101; C12N 2740/16222 20130101; A61K
2039/5256 20130101; A61P 37/04 20180101; A61P 33/00 20180101; C07K
14/005 20130101; A61K 2039/545 20130101; Y02A 50/30 20180101; A61K
39/015 20130101; A61K 2039/55583 20130101; A61P 31/04 20180101;
A61P 33/06 20180101; C12N 2740/16322 20130101 |
Class at
Publication: |
424/278.1 ;
424/204.1; 435/6; 435/7.1; 514/429 |
International
Class: |
C12Q 001/68; G01N
033/53; A61K 039/12; A61K 045/00; A61K 047/00; A61K 031/40; A01N
043/36 |
Goverment Interests
[0002] The research leading to the present invention was supported,
in part, by the grants AI-01682, AI-40656, and AI-47840 from the
National Institutes of Health. Accordingly, the U.S. government has
certain rights in the invention.
Claims
What is claimed is:
1. A method for augmenting the immunogenicity of an antigen in a
mammal, comprising immunizing the mammal with said antigen and
conjointly with an adjuvant comprising a glycosylceramide of the
general Formula 1: 17wherein R.sub.1, R.sub.2 and R.sub.5 represent
H or a specific monosaccharide; R.sub.3 and R.sub.6 represent H or
OH, respectively; R.sub.4 represents H, OH or a specific
monosaccharide; X denotes an integer from 1 to 23; R.sub.7
represents any one of the following groups (a)-(g): (a)
--(CH.sub.2).sub.11--CH.sub.3, (b) --(CH.sub.2).sub.12--CH.s- ub.3,
(c) --(CH.sub.2).sub.13--CH.sub.3, (d)
--(CH.sub.2).sub.9--CH(CH.sub- .3).sub.2, (e)
--(CH.sub.2).sub.10--CH(CH.sub.3).sub.2, (f)
--(CH.sub.2).sub.11--CH(CH.sub.3).sub.2, (g)
--(CH.sub.2).sub.11--CH(CH.s- ub.3)--C.sub.2H.sub.5.
2. The method of claim 1, wherein said glycosylceramide is selected
from the group consisting of .alpha.-galactosylceramide
(.alpha.-GalCer), .alpha.-glucosylceramide (.alpha.-GlcCer),
Gal.alpha.1-6Gal.alpha.1-1'Cer- , Gal.alpha.1-6Glc.alpha.1-1'Cer,
Gal.alpha.1-2Gal.alpha.1-1'Cer, and
Gal.beta.1-3Gal.alpha.1-1'Cer.
3. The method of claim 2, wherein said .alpha.-GalCer is
(2S,3S,4R)-1-O-(.alpha.-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-
-octadecanetriol.
4. The method of claim 1, wherein said antigen and said adjuvant
are administered simultaneously.
5. The method of claim 1, wherein said antigen is
malaria-specific.
6. The method of claim 5, wherein said malaria-specific antigen
comprises irradiated plasmodial sporozoites.
7. The method of claim 5, wherein said malaria-specific antigen
comprises a T cell epitope of the malarial circumsporozoite (CS)
protein.
8. The method of claim 1, wherein said antigen is HIV-specific.
9. The method of claim 1, wherein said antigen is presented by a
recombinant virus expressing said antigen.
10. The method of claim 9, wherein said virus is selected from the
group consisting of a recombinant adenovirus, recombinant pox
virus, and recombinant Sindbis virus.
11. The method of claim 1, wherein said mammal is human.
12. A method for enhancing or extending the duration of
antigen-specific Th1-type immune responses in a mammal comprising
conjointly administering to said mammal (i) an antigen and (ii) an
adjuvant comprising a glycosylceramide of the general Formula 1:
18wherein R.sub.1, R.sub.2 and R.sub.5 represent H or a specific
monosaccharide; R.sub.3 and R.sub.6 represent H or OH,
respectively; R.sub.4 represents H, OH or a specific
monosaccharide; X denotes an integer from 1 to 23; R.sub.7
represents any one of the following groups (a)-(g): (a)
--(CH.sub.2).sub.11--CH.sub.3, (b) --(CH.sub.2).sub.12--CH.sub.3,
(c) --(CH.sub.2).sub.13--CH.sub.3, (d)
--(CH.sub.2).sub.9--CH(CH.sub.3).sub.2, (e)
--(CH.sub.2).sub.10--CH(CH.su- b.3).sub.2, (f)
--(CH.sub.2).sub.11--CH(CH.sub.3).sub.2, (g)
--(CH.sub.2).sub.11--CH(CH.sub.3)--C.sub.2H.sub.5.
13. The method of claim 12, wherein said Th1-type immune responses
are CD8+ T cell responses.
14. A method for treating a disease in a mammal comprising
conjointly administering to said mammal an antigen and an adjuvant
comprising a glycosylceramide of the general Formula 1: 19wherein
R.sub.1, R.sub.2 and R.sub.5 represent H or a specific
monosaccharide; R.sub.3 and R.sub.6 represent H or OH,
respectively; R.sub.4 represents H, OH or a specific
monosaccharide; X denotes an integer from 1 to 23; R.sub.7
represents any one of the following groups (a)-(g): (a)
--(CH.sub.2).sub.11--CH.sub.3, (b) --(CH.sub.2).sub.12--CH.sub.3,
(c) --(CH.sub.2).sub.13--CH.sub.3, (d)
--(CH.sub.2).sub.9--CH(CH.sub.3).sub.2, (e)
--(CH.sub.2).sub.10--CH(CH.su- b.3).sub.2, (f)
--(CH.sub.2).sub.11--CH(CH.sub.3).sub.2, (g)
--(CH.sub.2).sub.11--CH(CH.sub.3)--C.sub.2H.sub.5.
15. The method of claim 14, wherein said glycosylceramide is
selected from the group consisting of .alpha.-galactosylcaramide
(.alpha.-GalCer), .alpha.-glucosylceramide (.alpha.-GlcCer),
Gal.alpha.1-6Gal.alpha.1-1'Cer- , Gal.alpha.1-6Glc.alpha.1-1'Cer,
Gal.alpha.1-2Gal.alpha.1-1'Cer, and
Gal.beta.1-3Gal.alpha.1-1'Cer.
16. The method of claim 15, wherein said .alpha.-GalCer is
(2S,3S,4R)-1-O-(.alpha.-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-
-octadecanetriol.
17. The method of claim 14, wherein said disease is selected from
the group consisting of infection and cancer.
18. The method of claim 17, wherein said infection is selected from
the group consisting of viral infection, bacterial infection,
parasitic infection, and fungal infection.
19. The method of claim 14, wherein said disease is malaria.
20. The method of claim 14, wherein said disease is HIV
infection.
21. The method of claim 14, wherein said mammal is human.
22. A pharmaceutical composition comprising an immunogenically
effective amount of an adjuvant comprising a glycosylceramide of
the general Formula 1: 20wherein R.sub.1, R.sub.2 and R.sub.5
represent H or a specific monosaccharide; R.sub.3 and R.sub.6
represent H or OH, respectively; R.sub.4 represents H, OH or a
specific monosaccharide; X denotes an integer from 1 to 23; R.sub.7
represents any one of the following groups (a)-(g): (a)
--(CH.sub.2).sub.11--CH.sub.3, (b) --(CH.sub.2).sub.12--CH.sub.3,
(c) --(CH.sub.2).sub.13--CH.sub.3, (d)
--(CH.sub.2).sub.9--CH(CH.sub.3).sub.2, (e)
--(CH.sub.2).sub.10--CH(CH.su- b.3).sub.2, (f)
--(CH.sub.2).sub.11--CH(CH.sub.3).sub.2, (g)
--(CH.sub.2).sub.11--CH(CH.sub.3)--C.sub.2H.sub.5.
23. The pharmaceutical composition of claim 22, wherein said
glycosylceramide is selected from the group consisting of
.alpha.-galactosylcaramide (.alpha.-GalCer),
.alpha.-glucosylceramide (.alpha.-GlcCer),
Gal.alpha.1-6Gal.alpha.1-1'Cer, Gal.alpha.1-6Glc.alpha.- 1-1'Cer,
Gal.alpha.1-2Gal.alpha.1-1'Cer, and Gal.beta.1-3Gal.alpha.1-1'Cer-
.
24. The pharmaceutical composition of claim 23, wherein said
.alpha.-GalCer is
(2S,3S,4R)-1-O-(.alpha.-D-galactopyranosyl)-2-(N-hexaco-
sanoylamino)- 1,3,4-octadecanetriol.
25. The pharmaceutical composition of claim 22 further comprising a
pharmaceutically acceptable carrier or excipient.
26. The pharmaceutical composition of claim 22 further comprising
an immunogenically effective amount of an antigen.
27. A method for augmenting the protective immunity induced by an
antigen in a mammal comprising administering to said mammal the
pharmaceutical composition of claim 22.
28. A method for treating a disease in a mammal comprising
administering to said mammal the pharmaceutical composition of
claim 22.
29. The method of claim 28, wherein said disease is selected from
the group consisting of infection and cancer.
30. The method of claim 29, wherein said infection is selected from
the group consisting of viral infection, bacterial infection,
parasitic infection, and fungal infection.
31. The method of claim 28, wherein said disease is malaria.
32. The method of claim 28, wherein said disease is HIV
infection.
33. A vaccine composition comprising an immunogenically effective
amount of an antigen and an immunogenically effective amount of an
adjuvant comprising a glycosylceramide of the general Formula 1:
21wherein R.sub.1, R.sub.2 and R.sub.5 represent H or a specific
monosaccharide; R.sub.3 and R.sub.6 represent H or OH,
respectively; R.sub.4 represents H, OH or a specific
monosaccharide; X denotes an integer from 1 to 23; R.sub.7
represents any one of the following groups (a)-(g): (a)
--(CH.sub.2).sub.11--CH.sub.3, (b) --(CH.sub.2).sub.12--CH.sub.3,
(c) --(CH.sub.2).sub.13--CH.sub.3, (d)
--(CH.sub.2).sub.9--CH(CH.sub.3).sub.2- , (e)
--(CH.sub.2).sub.10--CH(CH.sub.3).sub.2, (f)
--(CH.sub.2).sub.11--CH- (CH.sub.3).sub.2, (g)
--(CH.sub.2).sub.11--CH(CH.sub.3)--C.sub.2H.sub.5.
34. The vaccine composition of claim 33, wherein said
glycosylceramide is selected from the group consisting of
.alpha.-galactosylcaramide (.alpha.-GalCer),
.alpha.-glucosylceramide (.alpha.-GlcCer),
Gal.alpha.1-6Gal.alpha.1-1'Cer, Gal.alpha.1-6Glc.alpha.1-1'Cer,
Gal.alpha.1-2Gal.alpha.1-1'Cer, and
Gal.beta.1-3Gal.alpha.1-1'Cer.
35. The vaccine composition of claim 34, wherein said
.alpha.-GalCer is (2S,3S ,4R)- 1
-O-(.alpha.-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,-
3,4-octadecanetriol.
36. The vaccine composition of claim 33 further comprising a
pharmaceutically acceptable carrier or excipient.
37. A method for conferring immunity against the sporozoite stage
of malaria to a susceptible mammalian host comprising conjointly
administering to said host (i) at least one malaria-specific
antigen selected from the group consisting of sporozoite surface
antigens in a first amount, and (ii) .alpha.-galactosylcaramide
(.alpha.-GalCer) as an immune adjuvant in a second amount; said
first and second amounts being effective in combination to enhance
or prolong the immune response mounted against said antigen by the
host compared to the immune response that the host could have
mounted upon the administration of said first amount of said
antigen without the conjoint administration of said adjuvant.
38. The method of claim 37, wherein said .alpha.-GalCer is
(2S,3S,4R)-1-O-(.alpha.-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-
-octadecanetriol.
39. The method of claim 37, wherein said antigen and said adjuvant
are administered simultaneously.
40. The method of claim 37, wherein said malaria-specific antigen
comprises a T cell epitope of the plasmodial circumsporozoite (CS)
protein.
41. The method of claim 40, wherein said T cell epitope has an
amino acid sequence selected from the group consisting of
YNRNIVNRLLGDALNGKPEEK (SEQ ID NO: 1), SYVPSAEQI (SEQ ID NO: 2),
(NVDPNANP).sub.n (SEQ ID NO: 3), and EYLNKIQNSLSTEWSPC SVT (SEQ ID
NO: 4).
42. The method of claim 37, wherein said malaria-specific antigen
comprises a B cell epitope of the plasmodial circumsporozoite (CS)
protein.
43. The method of claim 42, wherein said B cell epitope has an
amino acid sequence (NANP).sub.3 (SEQ ID NO: 15).
44. The method of claim 37, wherein said malaria-specific antigen
is presented by a recombinant virus expressing said antigen.
45. The method of claim 44, wherein said virus is selected from the
group consisting of a recombinant adenovirus, recombinant pox
virus, and recombinant Sindbis virus.
46. The method of claim 37, wherein said host is human.
47. The method of claim 37, wherein said enhancement or extension
of the immune response is manifested by the enhancement or
extension of the duration of antigen-specific CD8+ T cell
responses.
48. The method of claim 37, wherein said first amount is in the
range of 0.1 .mu.g-100 mg per kg of body weight.
49. The method of claim 37, wherein said second amount is in the
range of 10-100 .mu.g per kg of body weight.
50. A method for enhancing a T cell response to an HIV antigen in a
susceptible mammalian host comprising conjointly administering to
said host: (i) at least one HIV-specific antigen selected from the
group consisting of Gag, Tat, Pol, Env, Nef, gp160, p18, and gp120
in a first amount, and (ii) .alpha.-galactosylcaramide
(.alpha.-GalCer) as an immune adjuvant in a second amount; said
first and second amounts being effective in combination to enhance
said T cell response mounted against said antigen by the host
compared to the immune response that the host could have mounted
upon the administration of said first amount of said antigen
without the conjoint administration of said adjuvant.
51. The method of claim 50, wherein said .alpha.-GalCer is
(2S,3S,4R)-1-O-(.alpha.-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-
-octadecanetriol.
52. The method of claim 50, wherein said HIV-specific antigen and
said adjuvant are administered concurrently.
53. The method of claim 50, wherein said adjuvant is administered
one hour prior to said antigen.
54. The method of claim 50, wherein said HIV-specific antigen
comprises a T cell epitope of the Gag, Tat, Env, Pol, Nef, gp160,
p18, or gp120.
55. The method of claim 54, wherein said T cell epitope has an
amino acid sequence selected from the group consisting of
RGPGRAFVTI (SEQ ID NO: 5), KAFSPEVIPMF (SEQ ID NO: 6), KAFSPEVI
(SEQ ID NO: 7), TPQDLNMML (SEQ ID NO: 8), TPQDLNTML (SEQ ID NO: 9),
DTINEEAAEW (SEQ ID NO: 10), KRWILGLNK (SEQ ID NO: 11), and
QATQEVKNW (SEQ ID NO: 12), RLRPGGKKK (SEQ ID NO: 13), and SLYNTVATL
(SEQ ID NO: 14).
56. The method of claim 50, wherein said HIV-specific antigen is
presented by a recombinant virus expressing said antigen.
57. The method of claim 50, wherein said virus is selected from the
group consisting of a recombinant adenovirus, recombinant pox
virus, and recombinant Sindbis virus.
58. The method of claim 50, wherein said host is human.
59. The method of claim 50, wherein said first amount is in the
range of 0.1 .mu.g-100 mg per kg of body weight.
60. The method of claim 50, wherein said second amount is in the
range of 10-100 .mu.g per kg of body weight.
61. A method for preparing a vaccine composition comprising at
least one antigen and an adjuvant comprising a glycosylceramide of
the general Formula 1: 22wherein R.sub.1, R.sub.2 and R.sub.5
represent H or a specific monosaccharide; R.sub.3 and R.sub.6
represent H or OH, respectively; R.sub.4 represents H, OH or a
specific monosaccharide; X denotes an integer from 1 to 23; R.sub.7
represents any one of the following groups (a)-(g): (a)
--(CH.sub.2).sub.11--CH.sub.3, (b) --(CH.sub.2).sub.12--CH.sub.3,
(c) --(CH.sub.2).sub.13--CH.sub.3, (d)
--(CH.sub.2).sub.9--CH(CH.sub.3).sub.2, (e)
--(CH.sub.2).sub.10--CH(CH.su- b.3).sub.2, (f)
--(CH.sub.2).sub.11--CH(CH.sub.3).sub.2, (g)
--(CH.sub.2).sub.11--CH(CH.sub.3)--C.sub.2H.sub.5, said method
comprising admixing the adjuvant and the antigen.
62. A kit for the preparation of a pharmaceutical or vaccine
composition comprising at least one antigen and an adjuvant,
wherein the adjuvant comprises a glycosylceramide of the general
Formula 1: 23wherein R.sub.1, R.sub.2 and R.sub.5 represent H or a
specific monosaccharide; R.sub.3 and R.sub.6 represent H or OH,
respectively; R.sub.4 represents H, OH or a specific
monosaccharide; X denotes an integer from 1 to 23; R.sub.7
represents any one of the following groups (a)-(g): (a)
--(CH.sub.2).sub.11--CH.sub.3, (b) --(CH.sub.2).sub.12--CH.sub.3,
(c) --(CH.sub.2).sub.13--CH.sub.3, (d)
--(CH.sub.2).sub.9--CH(CH.sub.3).sub.2- , (e)
--(CH.sub.2).sub.10--CH(CH.sub.3).sub.2, (f)
--(CH.sub.2).sub.11--CH- (CH.sub.3).sub.2, (g)
--(CH.sub.2).sub.11--CH(CH.sub.3)--C.sub.2H.sub.5, said kit
comprising the antigen in a first container, and the adjuvant in a
second container, and optionally instructions for admixing the
antigen and the adjuvant and/or for administration of the
composition; and wherein optionally the containers are in a
package.
63. The kit of claim 62 wherein the adjuvant is selected from the
group consisting of .alpha.-galactosylcaramide (.alpha.-GalCer),
.alpha.-glucosylceramide (.alpha.-GlcCer),
Gal.alpha.1-6Gal.alpha.1-1'Cer- , Gal.alpha.1-6Glc.alpha.1-1'Cer,
Gal.alpha.1-2Gal.alpha.1-1'Cer, and
Gal.beta.1-3Gal.alpha.1-1'Cer.
64. The kit of claim 63, wherein said .alpha.-GalCer is
(2S,3S,4R)-1-O-(.alpha.-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-
-octadecanetriol.
Description
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) from U.S. Provisional Patent Application Serial No.
60/308,056 filed Jul. 25, 2001, which is incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to the use of
glycosylceramides as adjuvants to augment the immunogenicity of
various infectious and tumor antigens.
BACKGROUND OF THE INVENTION
[0004] The successful elimination of pathogens, neoplastic cells,
or self-reactive immune mechanisms following prophylactic or
therapeutic immunization depends to a large extent on the ability
of the host's immune system to become activated in response to the
immunization and mount an effective response, preferably with
minimal injury to healthy tissue.
[0005] The rational design of vaccines initially involves
identification of immunological correlates of protection--the
immune effector mechanism(s) responsible for protection against
disease--and the subsequent selection of an antigen that is able to
elicit the desired adaptive response. Once this appropriate antigen
has been identified, it is essential to deliver it effectively to
the host's immune system.
[0006] In the design of effective vaccines, immunological adjuvants
serve as critical components, which accelerate, prolong, and/or
enhance an antigen-specific immune response as well as provide the
selective induction of the appropriate type of response.
[0007] New vaccines are presently under development and in testing
for the control of various neoplastic, autoimmune and infectious
diseases, including human immunodeficiency virus (HIV) and
tuberculosis. In contrast to older vaccines which were typically
based on live-attenuated or non-replicating inactivated pathogens,
modem vaccines are composed of synthetic, recombinant, or highly
purified subunit antigens. Subunit vaccines are designed to include
only the antigens required for protective immunization and are
believed to be safer than whole-inactivated or live-attenuated
vaccines. However, the purity of the subunit antigens and the
absence of the self-adjuvanting immunomodulatory components
associated with attenuated or killed vaccines often result in
weaker immunogenicity.
[0008] The immunogenicity of a relatively weak antigen can be
enhanced by the simultaneous or more generally conjoined
administration of the antigen with an "adjuvant", usually a
substance that is not immunogenic when administered alone, but will
evoke, increase and/or prolong an immune response to an antigen. In
the absence of adjuvant, reduced or no immune response may occur,
or worse the host may become tolerized to the antigen.
[0009] Adjuvants can be found in a group of structurally
heterogeneous compounds (Gupta et al., 1993, Vaccine, 11:293-306).
Classically recognized examples of adjuvants include oil emulsions
(e.g., Freund's adjuvant), saponins, aluminium or calcium salts
(e.g., alum), non-ionic block polymer surfactants,
lipopolysaccharides (LPS), mycobacteria, tetanus toxoid, and many
others. Theoretically, each molecule or substance that is able to
favor or amplify a particular situation in the cascade of
immunological events, ultimately leading to a more pronounced
immunological response can be defined as an adjuvant.
[0010] In principle, through the use of adjuvants in vaccine
formulations, one can (1) direct and optimize immune responses that
are appropriate or desirable for the vaccine; (2) enable mucosal
delivery of vaccines, i.e., administration that results in contact
of the vaccine with a mucosal surface such as buccal or gastric or
lung epithelium and the associated lymphoid tissue; (3) promote
cell-mediated immune responses; (4) enhance the immunogenicity of
weaker immunogens, such as highly purified or recombinant antigens;
(5) reduce the amount of antigen or the frequency of immunization
required to provide protective immunity; and (6) improve the
efficacy of vaccines in individuals with reduced or weakened immune
responses, such as newborns, the aged, and immunocompromised
vaccine recipients.
[0011] Although little is known about their mode of action, it is
currently believed that adjuvants augment immune responses by one
of the following mechanisms: (1) increasing the biological or
immunologic half-life of antigens (see, e.g., Lascelles, 1989, Vet.
Immunol. Immunopathol., 22: 15-27; Freund, 1956, Adv. Tuber. Res.,
7: 130-147); (2) improving antigen delivery to antigen-presenting
cells (APCs), as well as antigen processing and presentation by the
APCs (see, e.g., Fazekas de St. Groth et al., Immunol. Today, 19:
448-454, 1998), e.g., by enabling antigen to cross endosomal
membranes into the cytosol after ingestion of antigen-adjuvant
complexes by APCs (Kovacsovics-Bankowski et al., Science, 1995,
267: 243-246); (3) mimicking microbial structures leading to
improved recognition of microbially-derived antigens by the
pathogen-recognition receptors (PRRs), which are localized on
accessory cells from the innate immune system (Janeway, 1989, Cold
Spring Harbor Symp. Quant. Biol., 54:1-13; Medzhitov, 1997, Cell,
91:295-298; Rook, 1993, Immunol. Today, 14:95-96); (4) mimicking
danger-inducing signals from stressed or damaged cells which serve
to initiate an immune response (see, e.g., Matzinger, 1994, Annu.
Rev. Immunol., 12:991-209), (5) inducing the production of
immunomodulatory cytokines (see, e.g., Nohria, 1994, Biotherapy,
7:261-269; Iwasaki et al., 1997, J. Immunol., 158:4591-4601;
Maecker et al., 1997, Vaccine, 15:1687-1696); (6) biasing the
immune response towards a specific subset of the immune system
(e.g., generating Th1- or Th2-polarized response [see below], etc.)
(Janssen et al., Blood, 97:2758-2763, 2001; Yamamoto et al., Scand.
J. Immunol., 53:211-217, 2001; Weiner G. J., J. Leukoc. Biol.,
68:455-63, 2000; Lucey, Infect. Dis. Clin. North Am., 13:1-9,
1999), and (7) blocking rapid dispersal of the antigen challenge
(the "depot effect") (Hood et al., Immunology, Second Ed., 1984,
Benjamin/Cummings: Menlo Park, Calif.; St Clair et al., Proc. Natl.
Acad. Sci. U.S.A., 96:9469-9474, 1999; Ahao et al., J. Pharm. Sci.,
85:1261-1270, 1996; Morein et al., Vet. Immunol. Immunopathol.,
54:373-384, 1996). (See also reviews by Schijns, Curr. Opin.
Immunol., 12: 456-463, 2000; Vogel, Clin. Infect. Dis., 30 [Suppl.
3]: S266-70, 2000; Singh and O'Hagan, Nature Biotechnol., 17:
1075-81, 1999; Cox and Coulter, Vaccine, 15: 248-256, 1997).
[0012] Recent observations strongly suggest that endogenously
produced cytokines act as essential communication signals elicited
by traditional adjuvants. The redundancy of the cytokine network
makes it difficult to ascribe the activity of a particular adjuvant
to one or more cytokines. Cytokines crucial for immunogenicity may
include the proinflammatory (Type 1) substances: interferon
(IFN)-.alpha./.beta., tumor necrosis factor (TNF)-.alpha.,
interleukin (IL)-1, IL-6, IL-12, IL-15 and IL-18, which influence
antigen presentation. Others may act more downstream during clonal
expansion and differentiation of T and B cells, with IL-2, IL-4 and
IFN-.gamma. as prototypes (Brewer et al., 1996, Eur. J. Immunol.,
26:2062-2066; Smith et al., 1998, Immunology, 93:556-562).
Adjuvants that enhance immune responses through the induction of
IFN-.gamma. and delayed-type hypersensitivity also elicit the
production of IgG subclasses that are the most active in
complement-mediated lysis and in antibody-dependent
cell-mediated-cytotoxicity effector mechanisms (e.g., IgG2a in mice
and IgG1 in humans) (Allison, Dev. Biol. Stand., 1998, 92:3-11;
Unkeless, Annu. Rev. Immunol., 1988, 6:251-81; Phillips et al.,
Vaccine, 1992, 10:151-8).
[0013] Clearly, some adjuvants may perform more than one function.
For example, purified microbial components such as LPS or extracts
of Toxoplasma gondii rapidly increase not only the number of
antigen-presenting dendritic cells (DC) and their migration but
also IL-12 production (Souza et al., 1997, J. Exp. Med.,
186:1819-1829).
[0014] As different adjuvants may have diverse mechanisms of
action, their being chosen for use with a particular vaccine may be
based on the route of administration to be employed, the type of
immune responses desired (e.g., antibody-mediated, cell-mediated,
mucosal, etc.), and the particular inadequacy of the primary
antigen.
[0015] The benefit of incorporating adjuvants into vaccine
formulations to enhance immunogenicity must be weighed against the
risk that these agents will induce adverse local and/or systemic
reactions. Local adverse reactions include local inflammation at
the injection site and, rarely, the induction of granuloma or
sterile abscess formation. Systemic reactions to adjuvants observed
in laboratory animals include malaise, fever, adjuvant arthritis,
and anterior uveitis (Allison et al., Mol. Immunol., 1991,
28:279-84; Waters et al., Infect. Immun., 1986, 51:816-25). Such
reactions often are caused by the interaction of the adjuvant and
the antigen itself, or may be due to the type of response to a
particular antigen the adjuvant produces, or the cytokine profile
the adjuvant induces.
[0016] Thus, many potent immunoadjuvants, such as Freund's Complete
or Freund's Incomplete Adjuvant, are toxic and are therefore useful
only for animal research purposes, not human vaccinations.
Currently, aluminum salts and MF59 are the only vaccine adjuvants
approved for human use. Of the novel adjuvants under evaluation,
immunostimulatory molecules such as the lipopolysaccharide-derived
MPL and the saponin derivative QS-21 appear most promising,
although doubts have been raised as to their safety for human use.
Preclinical work with particulate adjuvants, such as the MF59
microemulsion and lipid-particle immuno-stimulating complexes
(ISCOMs), suggest that these molecules are also themselves potent
elicitors of humoral and cellular immune responses. In addition,
preclinical data on CpG oligonucleotides appear to be encouraging,
particularly with respect to their ability to manipulate immune
responses selectively. While all these adjuvants show promise, the
development of more potent novel adjuvants may allow novel vaccines
to be developed and both novel and existing vaccines to be used as
therapeutic as well as improved prophylactic agents.
[0017] Recently, a novel lymphoid lineage, natural killer T (NKT)
cells, distinct from mainstream T cells, B cells and NK cells, has
been identified (Arase et al., 1992, Proc. Natl Acad. Sci. USA,
89:6506; Bendelac et al., 1997, Annu. Rev. Immunol., 15:535). These
cells are characterized by co-expression of NK cell receptors and
semi-invariant T cell receptors (TCR) encoded by V.alpha.14 and
J.alpha.281 gene segments in mice and V.alpha.24 and J.alpha.Q gene
segments in humans. The activation of NKT cells in vivo promptly
induces a series of cellular activation events leading to the
activation of innate cells such as natural killer (NK) cells and
dendritic cells (DC), the activation of adaptive cells such as B
cells and T cells, the induction of co-stimulatory molecules and
the abrupt release of cytokines such as interleukin-4 (IL-4) and
interferon-.gamma. (IFN-.gamma.) (Burdin et al., Eur. J. Immunol.
29: 2014-2025, 1999; Carnaud et al., J. Immunol., 163: 4647-4650,
1999; Kitamura et al., J. Exp. Med., 189: 1121-1128, 1999; Kitamura
et al., Cell Immunol., 199: 37-42, 2000; Aderem and Ulevitch,
Nature, 406: 782-787, 2000). In addition, activated NKT cells can
themselves bring about killing mediated by Fas and perforin. The
full activation cascade can be recruited by the engagement of NKT
TCR. Alternatively, powerful T-helper-cell type 1 (Th1) functions
can be selectively triggered by cytokines such as interleukin-12
(IL-12) released by infected macrophages or DC. These functions are
believed likely to be correlated with the important role of NKT
cells in conditions such as autoimmune diabetes, rejection of
established tumours or the prevention of chemically induced tumours
(Yoshimoto et al, 1995, Science, 270: 1845; Hammond et al., J. Exp.
Med., 187: 1047-1056, 1998; Kawano et al., 1998, Proc. Natl. Acad.
Sci. USA, 95: 5690; Lehuen et al., J. Exp. Med., 188: 1831-1839,
1998; Wilson et al., Nature, 391: 177-181, 1998; Smyth et al., J.
Exp. Med., 191: 661-668, 2000). Finally, NKT cells are thought to
contribute to antimicrobial immunity through their capacity to
influence the Th1-Th2 polarization (Cui et al., J. Exp. Med., 190:
783-792, 1999; Singh et al., J. Immunol., 163: 2373-2377, 1999;
Shinkai and Locksley, J. Exp. Med., 191: 907-914, 2000). These
cells are therefore implicated as key effector cells in innate
immune responses. However, the potential role of NKT cells in the
development of adaptive immune responses remains unclear.
[0018] Recently, it was demonstrated that NKT cells can be
activated both in vitro and in vivo by .alpha.-galactosyl-ceramide
(.alpha.-GalCer), a glycolipid originally extracted from Okinawan
marine sponges (Natori et al., Tetrahedron, 50: 2771-2784, 1994),
or its synthetic analog KRN 7000 [(2S,3S,4R)-
1-O-(.alpha.-D-galactopyranosyl)-2-(N-hexacosanoylamino)-
1,3,4,-octadecanetriol] which can be obtained from Pharmaceutical
Research Laboratories, Kirin Brewery (Gumna, Japan) or synthesized
as described previously (see, e.g., Kobayashi et al., 1995, Oncol.
Res., 7:529-534; Kawano et al., 1997, Science, 278:1626-9; Burdin
et al., 1998, J. Immunol., 161:3271; Kitamura et al., 1999, J. Exp.
Med., 189:1121; U.S. Pat. No. 5,936,076). Thus, it was shown that
.alpha.-GalCer can stimulate NK activity and cytokine production by
NKT cells and exhibits potent antitumor activity in vivo (Kawano et
al., 1997, supra; Kawano et al., 1998, Proc. Natl Acad. Sci. USA,
95:5690; Kitamura et al., 1999, supra). Kitamura et al. (1999,
supra) demonstrated that the immunostimulating effect of
.alpha.-GalCer was initiated by CD40-CD40L-mediated NKT-DC
interactions. As the immunoregulatory functions of .alpha.-GalCer
were absent in both CD1d.sup.-1- and NKT-deficient mice, this
indicates that .alpha.-GalCer has to be presented by the MHC class
I-like molecule CD1d.
[0019] CD1 is a conserved family of non-polymorphic genes related
to MHC that seems to have evolved to present lipid and glycolipid
antigens to T cells and in this way participates in both an innate
and an adaptive pathway of antigen recognition (reviewed by Park
and Bendelac, Nature, 406: 788-792, 2000; see also Calabi et al.,
Eur. J. Immunol., 19: 285-292, 1989; Porcelli and Modlin, Annu.
Rev. Immunol., 17: 297-329, 1999). It comprises up to five distinct
genes (isotypes) that can be separated into two groups on the basis
of sequence homology. Group 1, which comprises CD1a, CD1b, CD1c and
CD1e, is present in humans but absent from mouse and rat. Group2,
which includes CD1d, is found in all species studied so far,
including humans.
[0020] CD1 isotypes are expressed selectively by antigen-presenting
cells such as dendritic cells (DCs), macrophages and subsets of B
cells, but apart from CD1d expression in hepatocytes they are
generally not expressed in solid tissues (Porcelli et al., supra;
Bendelac et al., Annu. Rev. Immunol., 15: 535-562, 1997).
[0021] .alpha.-GalCer is recognized in picomolar concentrations by
those among mouse and human CD1d-restricted lymphocytes that
express a semi-invariant TCR and exert potent effector and
regulatory functions (Kawano et al., Science, 278: 1626-1629,
1997). CD1d/.alpha.-GalCer complex is, in turn, recognized by the
antigen receptors of mouse V.alpha.14 and human V.alpha.24 natural
killer T (NKT) cells (Bendelac et al., Science, 268: 863-865, 1995;
Bendelac et al., Annu. Rev. Immunol., 15: 535-562, 1997; Park et
al., Eur. J. Immunol., 30: 620-625, 2000).
[0022] Upon binding to CD1d, .alpha.-GalCer was demonstrated to
activate murine NKT cells both in vivo and in vitro (Kawano et al.,
1997, Science, 278:1626-1629; Burdin et al., 1998, J. Immunol.,
161:3271-3281), and human NKT cells in vitro (Spada et al., 1998,
J. Exp. Med., 188:1529-1534; Brossay et al., 1998, J. Exp. Med.
188:1521-1528). For example, .alpha.-GalCer was shown to display
NKT-mediated anti-tumor activity in vitro by activating human NKT
cells (Kawano et al., 1999, Cancer Res., 59:5102-5105).
[0023] In addition to .alpha.-GalCer, other glycosylceramides
having .alpha.-anomeric conformation of sugar moiety and
3,4-hydroxyl groups of the phytosphingosine (such as
.alpha.-glucosylceramide [.alpha.-GlcCer],
Gal.alpha.1-6Gal.alpha.1-1'Cer, Gal.alpha.1-6Glc.alpha.1-1'Cer,
Gal.alpha.1-2Gal.alpha.1-1'Cer, and Gal.beta.1-3Gal.alpha.1-1'Cer)
have been demonstrated to stimulate proliferation of V.alpha.14 NKT
cells in mice, although with lower efficiency (Kawano et al.,
Science, 278: 1626-1629, 1997). By testing a panel of
.alpha.-GalCer analogs for reactivity with mouse V.alpha.14 NKT
cell hybridomas, Brossay et al. (J. Immunol., 161: 5124-5128, 1998)
determined that nearly complete truncation of the .alpha.-GalCer
acyl chain from 24 to 2 carbons does not significantly affect the
mouse NKT cell response to glycolipid presented by either mouse CD1
or its human homolog.
[0024] It has been also demonstrated that in vivo administration
.alpha.-GalCer not only causes the activation of NKT cells to
induce a strong NK activity and cytokine production (e.g., IL-4,
IL-12 and IFN-.gamma.) by CD1d-restricted mechanisms, but also
induces the activation of immunoregulatory cells involved in
acquired immunity (Nishimura et al., 2000, Int. Immunol., 12:
987-994). Specifically, in addition to the activation of
macrophages and NKT cells, it was shown that in vivo administration
of .alpha.-GalCer resulted in the induction of the early activation
marker CD69 on CD4.sup.+ T cells, CD8.sup.+ T cells, and B cells
(Burdin et al., 1999, Eur. J. Immunol. 29: 2014; Singh et al.,
1999, J. Immunol. 163: 2373; Kitamura et al., 2000, Cell. Immunol.
199:37; Schofield et al., 1999, Science 283: 225; Eberl et al.,
2000, J. Immunol., 165:4305-4311). These studies open the
possibility that .alpha.-GalCer may play an equally important role
in bridging not only innate immunity mediated by NKT cells, but
also adaptive immunity mediated by B cells, T helper (Th) cells and
T cytotoxic (Tc) cells.
[0025] Due to the identification of new tumor-specific antigens and
realization that the immune system plays a critical role in the
prevention of cancer and the control of tumor growth, in recent
years, there has been a renewed interest in the development of
therapeutic cancer vaccines (e.g., to reduce tumor burden and
control metastasis).
[0026] The demonstration that in vivo engagement of NKT cells by
their glycolipid ligand .alpha.-GalCer rapidly induces a cascade of
cellular activation that involves elements common to innate and
adaptive immunity as well as the generation of tumor-specific
cytotoxic T cells (Nishimura et al., 2000, supra) suggests that
.alpha.-GalCer administration may generally affect not only the
speed and strength but also the type of subsequent immune
responses, in particular, those directed against tumor cells.
Indeed, Kabayashi et al. (1995, Oncol. Res., 7: 529-534) discovered
that a synthetic form of .alpha.-GalCer (KRN 7000) had stronger
antimetastatic activities in B 16-bearing mice than biological
response modifiers such as OK432 and Lentinan and a
chemotherapeutic agent Mitomycin C. In these experiments, 60% of
mice bearing tumors were cured by treatment with 100 .mu.g/kg
KRN7000. KRN7000 was also shown to induce a pronounced
tumor-specific immunity in mice with liver metastasis of murine
T-lymphoma EL-4 cells (Nakagawa et al., Oncol. Res., 10: 561-568,
1998) or Colon26 cells (Nakagawa et al., Cancer Res., 58:
1202-1207, 1998). Furthermore, the administration of .alpha.-GalCer
to mice was found to inhibit the development of hepatic metastasis
of primary melanomas (Kawano et al., 1998, Proc. Natl. Acad. Sci.
USA, 95: 5690-5693).
[0027] The data presented above have led the present inventors to a
hypothesis that the glycosylceramide-induced NKT cell responses may
also contribute to immune responses involved in combating various
infections. Indeed, the present inventors and co-workers have
recently observed that the administration of .alpha.-GalCer to mice
resulted rapidly in strong anti-malaria activity, inhibiting the
development of intra-hepatocytic stages of the rodent malaria
parasites, P. yoeli and P. berghei (Gonzalez-Aseguinolaza et al.,
2000, Proc. Natl. Acad. Sci. USA, 97: 8461-8466). The
administration of .alpha.-GalCer alone to mice lacking either CD1d
or V.alpha.14 NKT cells, however, failed to protect them against
malaria, indicating that the anti-malaria activity of
.alpha.-GalCer requires both NKT cells and the expression of CD1d.
Furthermore, .alpha.-GalCer was unable to inhibit parasite
development in the liver of mice lacking either IFN-.gamma. or the
IFN-.gamma. receptor, indicating that the anti-malaria activity of
the glycolipid is primarily mediated by IFN-.gamma..
[0028] In light of the data on the NKT-mediated anti-tumor and
anti-parasite activity of .alpha.-GalCer, it has been proposed that
this glycolipid is a potent inducer of protective immune responses
(see, e.g., Park and Bendelac, supra). The present inventors have
significantly expanded these hypotheses by conceiving and
demonstrating for the first time that .alpha.-GalCer and related
glycosylceramides can be employed not just as antigens but also as
adjuvants capable of enhancing and/or extending the duration of the
protective immune responses induced by other antigens. This is an
unexpected discovery, because .alpha.-GalCer-mediated NKT cell
activation results in the complete elimination of malaria-infected
cells, thus eliminating the source of antigen necessary for the
development of an adaptive immune response. In fact, the
administration of .alpha.-GalCer two days before immunization with
irradiated sporozoites almost completely abolishes
sporozoites-induced protection. Therefore, in order to use
.alpha.-GalCer as an adjuvant, the timing of the administration in
relation to the antigen given is very important.
[0029] Accordingly, the present invention provides for the first
time methods and compositions for enhancing and/or extending the
duration of the immune response against an antigen in a mammal,
notably a human, involving the conjoint immunization of the mammal
with (i) an antigen and (ii) an adjuvant comprising
glycosylceramide, in particular, .alpha.-GalCer.
[0030] Importantly, in addition to its ability to stimulate immune
responses, it has been demonstrated that .alpha.-GalCer,
independently of its dosage, does not induce toxicity in rodents
and monkeys (Nakagawa et al., 1998, Cancer Res., 58: 1202-1207).
Moreover, although a recent study showed the transient elevation of
liver enzyme activities immediately after .alpha.-GalCer treatment
in mice, suggesting a minor liver injury (Osman et al., 2000, Eur.
J. Immunol., 39: 1919-1928), human trials are currently being
conducted using .alpha.-GalCer to treat cancer patients without a
notable complication (Giaccone et al., 2000, Abstract. Proc. Amer.
Soc. Clin. Oncol., 19: 477a). Finally, unlike many other newly
developed adjuvants (see below), .alpha.-GalCer and related
glycosylceramides can be produced synthetically with reasonable
yields and efficiency (see, e.g., U.S. Pat. No. 5,936,076). All of
these factors make glycosylceramides and, in particular
.alpha.-GalCer, desirable adjuvant candidates.
[0031] In contrast to .alpha.-GalCer and related glycosylceramides,
conventional vaccine delivery systems and the adjuvants approved
for human use, aluminium salts and MF59 (Singh and O'Hagan, Nat.
Biotechnol., 17: 1075-1081, 1999), are poor at inducing CD8+ T cell
responses. Although certain novel adjuvants, such as purified
saponins, immunostimulatory complexes, liposomes, CpG DNA motifs,
and recombinant attenuated viruses (e.g., adenovirus, Sindbis
virus, influenza virus, and vaccinia virus), have been shown to
improve the antigen specific cellular immune responses over those
induced by the same antigen given alone or in combination with
standard alum adjuvants (Newman et al., J. Immunol., 1992;
148:2357-2362; Takahashi et al., Nature, 1990, 344:873-875; Babu et
al., Vaccine, 1995, 13:1669-1676; Powers et al., J. Infect. Dis.,
1995, 172:1103-7; White et al., Vaccine, 1995, 13:1111-1122; Krieg
et al., Trends Microbiol., 6: 23-27, 1998; Rodrigues et al., J.
Immunol., 158: 1268-1274, 1997; Tsuji et al., J. Virol., 72:
6907-6910, 1998; Li et al., Proc. Natl. Acad. Sci. USA, 90:
5214-52188, 1993), none of the currently available adjuvants
combine low toxicity in humans, cost-efficiency of production and
the ability to efficiently stimulate the immune system.
[0032] The development of an adaptive immune response is a
multifactorial phenomenon, in which many elements participate. In
this regard, .alpha.-GalCer-activated NKT cells induce the
activation of many of the elements involved in the development of
the adaptive immune response, such as antigen presenting cells
(APC), B cells, T helper (Th) cells and T cytotoxic (Tc) cells.
Therefore, theoretically, .alpha.-GalCer could be an ideal
immunomodulator. Additional advantage is that .alpha.-GalCer can be
administered and activate the immune system via many different
routes, including oral, subcutaneous, and intramuscular routes,
which are suitable for human use. Finally, it has been shown that
.alpha.-GalCer does not induce toxicity in rodents and monkeys
(Nakagawa et al., Cancer Res., 58:1202-1207, 1998).
[0033] Accordingly, there is a great need in the art to develop new
adjuvants that would combine low toxicity and easy availability
with the ability to enhance and/or prolong the antigen-specific
immune responses to a significant degree. The present invention
addresses these and other needs in the art by providing
glycosylceramides, a novel group of adjuvants with superior
properties. Such adjuvants can improve prophylactic and/or
therapeutic vaccines for the treatment of various infections and
cancers.
SUMMARY OF THE INVENTION
[0034] An object of the present invention is to provide a method
for augmenting an immunogenicity of an antigen in a mammal,
comprising administering said antigen conjointly with an adjuvant
composition comprising a glycosylceramide of the general Formula 1:
1
[0035] wherein R.sub.1, R.sub.2 and R.sub.5 represent H or a
specific monosaccharide; R.sub.3 and R.sub.6 represent H or OH,
respectively; R.sub.4 represents H, OH or a specific
monosaccharide; X denotes an integer from 1 to 23; R.sub.7
represents any one of the following groups (a)-(g):
(a)--(CH.sub.2).sub.11--CH.sub.3, (b) --(CH.sub.2).sub.12--CH.su-
b.3, (c) --(CH.sub.2).sub.13--CH.sub.3, (d)
--(CH.sub.2).sub.9--CH(CH.sub.- 3).sub.2, (e)
--(CH.sub.2).sub.10--CH(CH.sub.3).sub.2, (f)
--(CH.sub.2).sub.11--CH(CH.sub.3).sub.2, (g)
--(CH.sub.2).sub.11--CH(CH.s- ub.3)--C.sub.2H.sub.5.
[0036] A preferred adjuvant of the present invention comprises
.alpha.-galactosylceramide .alpha.-GalCer), specifically,
(2S,3S,4R)-1-O-(.alpha.-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-
-octadecanetriol represented by the Formula 2: 2
[0037] According to the present invention, the use of
glycosylceramide as an adjuvant results in an enhancement and/or
extension of the duration of the protective immunity induced by the
antigen and is attributed at least in part to the enhancement
and/or extension of antigen specific Th1-type responses, in
particular, CD8+ T cell responses.
[0038] The glycosylceramide-containing adjuvant of the invention
can be conjointly administered with any antigen, in particular,
with antigens derived from infectious agents or tumors. Preferably,
the adjuvant and antigen are administered simultaneously, most
preferably in a single dosage form.
[0039] In a further embodiment, the invention provides a
prophylactic and/or therapeutic method for treating a disease in a
mammal comprising administering to said mammal an immunoprotective
antigen together with an adjuvant composition that includes
glycosyl-ceramide. As specified herein, this method can be useful
for preventing and/or treating various infectious or neoplastic
diseases. In a preferred embodiment, the method of the invention is
employed to treat an infection selected from the group consisting
of viral infection, bacterial infection, parasitic infection, and
fungal infection.
[0040] Thus, in a specific embodiment, the present invention
discloses a method for conferring immunity against the sporozoite
stage of malaria in a mammal (e.g., human), wherein said method
comprises conjointly administering to said mammal a
malaria-specific antigen and an immunoadjuvant comprising
.alpha.-GalCer. In another specific embodiment, the invention
discloses a method for enhancing the immune response to HIV
infection (and potentially preventing and/or treating AIDS) in a
mammal, wherein said method comprises conjointly administering to
said mammal an HIV-specific antigen and an adjuvant comprising
.alpha.-GalCer. Additional specific methods disclosed herein
include without limitation:
[0041] (i) enhancing the immune response to Mycobacterium bovis
Bacillus Calmette-Gurin for prevention of M. tuberculosis
infection, by administering Mycobacterium bovis Bacillus
Calmette-Gurin and an adjuvant comprising .alpha.-GalCer;
[0042] (ii) enhancing the immune response to melanoma by
administering a plasmid cDNA coding for the human
melanoma-associated antigen, gp100, and an adjuvant comprising
.alpha.-GalCer;
[0043] (iii) enhancing the immune response to Candida albicans by
administering peptides derived from the immunodominant antigen, 65
kDa mannoprotein (MP65) and an adjuvant comprising
.alpha.-GalCer.
[0044] In conjunction with the methods of the present invention,
also provided are pharmaceutical and vaccine compositions
comprising an immunogenically effective amount of an antigen and an
immunogenically effective amount of an adjuvant selected from
glycosyl-ceramides within Formula 1 as well as, optionally, a
pharmaceutically acceptable carrier or excipient. In a specific
embodiment, glycosylceramide used for the preparation of the
adjuvant of the invention is .alpha.-GalCer, specifically
[(2S,3S,4R)-1-O-(.alpha.-D-galactopyranosyl)-2-(N-hexacosano-
ylamino)- 1,3,4-octadecanetriol].
[0045] The antigens useful in the compositions of the present
invention include without limitation various viral, bacterial,
fungal, parasite-specific, and tumor-specific antigens.
Non-limiting examples of viral antigens of the invention include
HIV antigens such as gp120, gp160, p18, Tat, Gag, Pol, Env, Nef;
glycoprotein from Herpesvirus; surface antigen and core antigen
from Hepatitis B virus. Non-limiting examples of bacterial antigens
of the invention include OspA, OspB and OspC antigens from Borrelia
sp. Non-limiting examples of fungal and parasite antigens of the
invention include MP65 from Candida albicans and CS protein from
Plasmodium sp., respectively. Non-limiting examples of
tumor-specific antigens of the invention include Melan A and gp100
antigens from melanoma.
[0046] In a specific embodiment, the antigen is malaria-specific
and comprises, for example, irradiated plasmodial sporozoites or a
synthetic peptide antigen comprising a T cell epitope of the
malarial circumsporozoite (CS) protein such as CD4+ T cell epitope
YNRNIVNR LLGDALNGKPEEK (SEQ ID NO: 1) or CD8+ T cell epitope
SYVPSAEQI (SEQ ID NO: 2) of P. yoelii CS protein, or CD4+ T cell
epitope (NVDPNANP).sub.n (SEQ ID NO: 3), or CD4+/CD8+ T cell
epitope EYLNKIQNSLSTEWSPCSVT (SEQ ID NO: 4) of P. falciparum CS
protein. In another preferred embodiment, the antigen is
HIV-specific such as CD8+ T cell epitope RGPGRAFVTI (SEQ ID NO: 5)
of p18 protein or HIV-1 Gag p24 CD8+ T cell epitopes (e.g.,
KAFSPEVIPMF (aa 30-40, SEQ ID NO: 6), KAFSPEVI (aa 30-37, SEQ ID
NO: 7), TPQDLNM (or T) ML (aa 180-188, SEQ ID NOS: 8 and 9),
DTINEEAAEW (aa 203-212, SEQ ID NO: 10), KRWIILGLNK (aa 263-272, SEQ
ID NO: 11), and QATQEVKNW (aa 308-316, SEQ ID NO: 12)), or Gag p17
CD8+ T cell epitopes (e.g., RLRPGGKKK (aa 20-29, SEQ ID NO: 13) and
SLYNTVATL (aa 77-85, SEQ ID NO: 14)).
[0047] In a specific embodiment, the antigen is presented by a
recombinant virus expressing said antigen. Preferably, the virus is
selected from the group consisting of a recombinant adenovirus,
recombinant pox virus, and recombinant Sindbis virus.
[0048] The invention also provides a method for preparing a vaccine
composition comprising at least one antigen and a
glycosylceramide-contai- ning adjuvant, said method comprising
admixing the adjuvant and the antigen.
[0049] In a related embodiment, the present invention provides a
kit for the preparation of a pharmaceutical or vaccine composition
comprising at least one antigen and a glycosylceramide-containing
adjuvant, said kit comprising the antigen in a first container, and
the adjuvant in a second container, and optionally instructions for
admixing the antigen and the adjuvant and/or for administration of
the composition; and wherein optionally the containers are in a
package.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIGS. 1A-D. .alpha.-GalCer enhances protective anti-malaria
immunity induced by irradiated sporozoites and recombinant viruses
expressing a plasmodial antigen. A. Groups of BALB/c mice were
co-injected intraperitoneally with different doses of
.alpha.-GalCer (0.5, 1, or 2 .mu.g) or vehicle (-), together with
intravenous immunization with P. yoelii irradiated sporozoites
(.gamma.-spz). Two weeks later all groups of mice were challenged
with infective sporozoites, and the amount of parasite rRNA in the
livers was measured by real time RT-PCR. Sera from immunized and
non-immunized mice were collected and their titers of
anti-sporozoite antibodies assayed by IFA. B. A single dose of
.alpha.-GalCer was administered two days before (-2), the same day
(0) or two days after (+2) immunization with (.gamma.-spz into
BALB/c (.box-solid.) or B10.D2 (.quadrature.) mice. C. A group of
BALB/c mice was immunized subcutaneously with a recombinant
adenovirus expressing the P. yoelii CS protein, AdPyCS, together
with s.c. administration of .alpha.-GalCer (+) or vehicle (-). D. A
group of BALB/c mice was immunized s.c. with a recombinant Sindbis
virus expressing a CD8+T cell epitope of the P. yoelii CS protein,
SIN(Mal), together with s.c. administration of .alpha.-GalCer (+)
or vehicle (-). Asterisk (*) indicates a significant (P<0.01)
difference between the two values using an unpaired t-test. In B-D,
all groups of mice were infected with live P. yoelii sporozoites
two weeks later, and the parasite burden in the liver was
determined as described in A. Results are expressed as the mean
values.+-.SD of five mice.
[0051] FIGS. 2A-C. .alpha.-GalCer increases the level of
antigen-specific T cell responses elicited by various vaccines. A.
A group of BALB/c mice was immunized s.c. with .gamma.-spz together
with or without administration of .alpha.-GalCer by the same route,
and two or six weeks later splenic lymphocytes were isolated and
the number of IFN-.gamma. secreting CS-specific CD8+ (.box-solid.)
and CD4+ (.quadrature.) T cells was determined by an ELISPOT assay.
B. A group of BALB/c mice was immunized s.c. with a recombinant
adenovirus expressing the P. yoelii CS protein, AdPyCS, together
with s.c. administration of .alpha.-GalCer (+) or vehicle (-). Two
weeks later the number of IFN-.gamma. secreting CS-specific
CD8+(.box-solid.) and CD4+ (.quadrature.) T cells was determined by
an ELISPOT assay. C. A group of BALB/c mice was immunized s.c. with
a recombinant Sindbis virus expressing a CD8+ T cell epitope of the
P. yoelii CS protein, SIN(Mal), or a recombinant Sindbis virus
expressing a CD8+ T cell epitope of p18 protein of HIV, SIN(p18),
together with s.c. administration of .alpha.-GalCer (+) or vehicle
(-). Two weeks later the number of IFN-.gamma. secreting
CS-specific and p18-specific CD8+ T cells was determined by an
ELISPOT assay. The data represent one of two experiments with
similar results and are expressed as the mean values.+-.SD of three
mice.
[0052] FIGS. 3A and 3B. .alpha.-GalCer prolongs the duration of the
protective anti-malaria immune responses elicited by .gamma.-spz.
A. BALB/c mice were immunized with .gamma.-spz together with
.alpha.-GalCer (+) or vehicle (-), as in FIG. 2, and two to four
weeks later the number of IFN-.gamma. secreting CS-specific CD8+T
cells in the spleens was determined by an ELISPOT assay. B. BALB/c
mice treated with .alpha.-GalCer (+) or vehicle (-) were immunized
with either 1.times.10.sup.4 or 1.times.10.sup.5 .gamma.-spz, and
two or four weeks later respectively, these plus non-immunized mice
were challenged with 50 viable P. yoelii sporozoites. Occurrence of
blood infection was determined by monitoring parasitemia in thin
blood smears from days 3 to 14 after the challenge.
[0053] FIGS. 4A and 4B. The adjuvant activity of .alpha.-GalCer
requires CD1d molecules and V.alpha.14 NKT cells. A. Groups of
CD1d-deficient (CD1.sup.-/-), V.alpha.14 NKT (J.alpha.281.sup.-/-)
deficient and wild-type (WT) mice on a BALB/c background were
immunized i.v. with .gamma.-spz together with i.p. administration
of .alpha.-GalCer (+) or vehicle (-). Two weeks later these and
non-immunized mice were challenged with viable sporozoites, and the
parasite burden in the liver was measured as described in FIG. 1.
B. Identical groups of mice as described in A were immunized with
.gamma.-spz with i.p. injection of .alpha.-GalCer (+) or vehicle
(-). Two weeks later the number of IFN-.gamma. secreting
CS-specific CD8+ T cells in the spleens was determined by an
ELISPOT assay. Asterisk (*) indicates a significant (P<0.01)
difference between the two values using an unpaired t-test. The
results reflect two experiments with similar results and are
expressed as the mean values.+-.SD of five (A) or three (B)
mice.
[0054] FIGS. 5A-C. The adjuvant activity of .alpha.-GalCer is
abolished in IFN-.gamma. receptor-deficient mice. A. Groups of
IFN-.gamma. receptor-deficient (IFN-.gamma.R.sup.-/-) and wild-type
(WT) mice on a B10.D2 background were immunized i.v. with
.gamma.-spz together with i.p. administration of .alpha.-GalCer (+)
or vehicle (-). Two weeks later splenic lymphocytes were obtained
and the number of IFN-.gamma. secreting CS-specific CD8+
(.box-solid.) and CD4+ (.quadrature.) T cells were determined by an
ELISPOT assay. B. Hepatic lymphocytes were obtained from
IFN-.gamma.R.sup.-/- and WT mice and stained with PE-labeled
CD1d/.alpha.-GalCer tetramer and FITC-labeled anti-CD3 antibody,
and the percentage of .alpha.-GalCer-specific T cells was
determined by flow cytometric analysis. The number indicated in the
upper right comers represents the percentage of double-positive
cells among the liver lymphoid cell population. C. Hepatic
lymphocytes were obtained from IFN-.gamma.R.sup.-/- (.box-solid.)
or WT (.quadrature.) mice, and the number of IFN-.gamma. or IL-4
secreting .alpha.-GalCer-specific cells were determined by an
ELISPOT assay. Results are expressed as the mean values.+-.SD of
five mice.
DETAILED DESCRIPTION OF THE INVENTION
[0055] Definitions
[0056] The terms "adjuvant" and "immunoadjuvant" are used
interchangeably in the present invention and refer to a compound or
mixture that may be non-immunogenic when administered to a host
alone, but that augments the host's immune response to another
antigen when administered conjointly with that antigen.
[0057] Adjuvant-mediated enhancement and/or extension of the
duration of the immune response can be assessed by any method known
in the art including without limitation one or more of the
following: (i) an increase in the number of antibodies produced in
response to immunization with the adjuvant/antigen combination
versus those produced in response to immunization with the antigen
alone; (ii) an increase in the number of T cells recognizing the
antigen or the adjuvant; and (iii) an increase in the level of one
or more Type I cytokines.
[0058] Adjuvants of the invention comprise compounds which belong
to the class of sphingoglycolipids, specifically glycosylceramides,
which can be represented by a general Formula 1: 3
[0059] wherein R.sub.1, R.sub.2 and R.sub.5 represent H or a
specific monosaccharide; R.sub.3 and R.sub.6 represent H or OH,
respectively; R.sub.4 represents H, OH or a specific
monosaccharide; X denotes an integer from 1 to 23; R.sub.7
represents any one of the following groups (a)-(g): (a)
--(CH.sub.2).sub.11--CH.sub.3, (b) --(CH.sub.2).sub.12--CH.s- ub.3,
(c) --(CH.sub.2).sub.13--CH.sub.3, (d)
--(CH.sub.2).sub.9--CH(CH.sub- .3).sub.2, (e)
--(CH.sub.2).sub.10--CH(CH.sub.3).sub.2, (f)
--(CH.sub.2).sub.11--CH(CH.sub.3).sub.2, (g)
--(CH.sub.2).sub.11--CH(CH.s- ub.3)--C.sub.2H.sub.5.
[0060] A preferred adjuvant of the present invention comprises
.alpha.-galactosylceramide (.alpha.-GalCer), specifically,
(2S,3S,4R)-1-O-(.alpha.-D-galactopyranosyl)-2-(N-hexacosanoylamino)-
1,3,4-octadecanetriol represented by the Formula 2: 4
[0061] Other examples of glycosylceramides useful in adjuvants of
the present invention include, without limitation:
[0062] .alpha.-glucosylceramide (.alpha.-GlcCer), specifically
(2S,3S,4R)-1-O-(.alpha.-D-glucopyranosyl)-N-hexacosanoyl-2-amino-
1,3,4-octadecanetriol, of the Formula 3: 5
[0063] Gal.alpha.1-6Gal.alpha.1-1'Cer, specifically
(2S,3S,4R)-2-amino- 1-O-(.alpha.-D-galactopyranosyl-(
1-6)-.alpha.-D-galactopyranosyl)-N-hexa-
cosanoyl-1,3,4-octadecanetriol, of the Formula 4: 6
[0064] Gal.alpha.1-6Glc.alpha.1-1'Cer, specifically
(2S,3S,4R)-2-amino-1-O-(.alpha.-D-galactopyranosyl-(1-6)-.alpha.-D-glucop-
yranosyl)-N-hexacosanoyl-1,3,4-octadecanetriol, of the Formula 5:
7
[0065] Gal.alpha.1-2Glc.alpha.1-1'Cer, specifically
(2S,3S,4R)-2-amino-1-O-(.alpha.-D-glucopyranosyl-(1-2)-.alpha.-D-galactop-
yranosyl)-N-[(R)-2-hydroxytetracosanoyl]-1,3,4-octadecanetriol, of
the Formula 6: 8
[0066] Gal.beta.1-3Gal.alpha.1-1'Cer, specifically
(2S,3S,4R)-2-amino-
1-O-(.beta.-D-galactofuranosyl-(1-4)-.alpha.-D-galactopyranosyl)-N-[(R)-2-
-hydroxytetracosanoyl]-1,3,4-octadecanetriol, of the Formula 7:
9
[0067] Preferably, the adjuvant of the invention is
pharmaceutically acceptable for use in humans.
[0068] The adjuvant of the invention can be administered as part of
a pharmaceutical or vaccine composition comprising an antigen or as
a separate formulation, which is administered conjointly with a
second composition containing an antigen. In any of these
compositions glycosylceramide can be combined with other adjuvants
and/or excipients/carriers. These other adjuvants include, but are
not limited to, oil-emulsion and emulsifier-based adjuvants such as
complete Freund's adjuvant, incomplete Freund's adjuvant, MF59, or
SAF; mineral gels such as aluminum hydroxide (alum), aluminum
phosphate or calcium phosphate; microbially-derived adjuvants such
as cholera toxin (CT), pertussis toxin, Escherichia coli
heat-labile toxin (LT), mutant toxins (e.g., LTK63 or LTR72),
Bacille Calmette-Guerin (BCG), Corynebacterium parvum, DNA CpG
motifs, muramyl dipeptide, or monophosphoryl lipid A; particulate
adjuvants such as immunostimulatory complexes (ISCOMs), liposomes,
biodegradable microspheres, or saponins (e.g., QS-21); cytokines
such as IFN-.gamma., IL-2, IL-12 or GM-CSF; synthetic adjuvants
such as nonionic block copolymers, muramyl peptide analogues (e.g.,
N-acetyl-muramyl-L-threonyl-D-isoglutamine [thr-MDP],
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,
N-acetylmuramyl-L-alanyl-D--
isoglutaminyl-L-alanine-2-[1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphory-
loxy]-ethylamine), polyphosphazenes, or synthetic polynucleotides,
and surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, hydrocarbon emulsions, or keyhole
limpet hemocyanins (KLH). Preferably, these additional adjuvants
are also pharmaceutically acceptable for use in humans.
[0069] Within the meaning of the present invention, the term
"conjoint administration" is used to refer to administration of an
immune adjuvant and an antigen simultaneously in one composition,
or simultaneously in different compositions, or sequentially. For
the sequential administration to be considered "conjoint", however,
the antigen and adjuvant must be administered separated by a time
interval that still permits the adjuvant to augment the immune
response to the antigen. For example, when the antigen is a
polypeptide, the antigen and adjuvant are administered on the same
day, preferably within an hour of each other, and most preferably
simultaneously. However, when nucleic acid is delivered to the
subject and the polypeptide antigen is expressed in the subject's
cells, the adjuvant is administered within 24 hours of nucleic acid
administration, preferably within 6 hours.
[0070] As used herein, the term "immunogenic" means that an agent
is capable of eliciting a humoral or cellular immune response, and
preferably both. An immunogenic entity is also antigenic. An
immunogenic composition is a composition that elicits a humoral or
cellular immune response, or both, when administered to an animal
having an immune system.
[0071] The term "antigen" refers to any agent (e.g., protein,
peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, or
combination thereof) that, when introduced into a host, animal or
human, having an immune system (directly or upon expression as in,
e.g., DNA vaccines), is recognized by the immune system of the host
and is capable of eliciting an immune response. As defined herein,
the antigen-induced immune response can be humoral or
cell-mediated, or both. An agent is termed "antigenic" when it is
capable of specifically interacting with an antigen recognition
molecule of the immune system, such as an immunoglobulin (antibody)
or T cell antigen receptor (TCR). Within the meaning of the present
invention, the antigens are preferably "surface antigens", i.e.,
expressed naturally on the surface of a pathogen, or the surface of
an infected cell, or the surface of a tumor cell. A molecule that
is antigenic need not be itself immunogenic, i.e., capable of
eliciting an immune response without an adjuvant or carrier.
[0072] The term "epitope" or "antigenic determinant" refers to any
portion of an antigen recognized either by B cells, or T cells, or
both. Preferably, interaction of such epitope with an antigen
recognition site of an immunoglobulin (antibody) or T cell antigen
receptor (TCR) leads to the induction of antigen-specific immune
response. T cells recognize proteins only when they have been
cleaved into smaller peptides and are presented in a complex called
the "major histocompatability complex (MHC)" located on another
cell's surface. There are two classes of MHC complexes-class I and
class II, and each class is made up of many different alleles.
Class I MHC complexes are found on virtually every cell and present
peptides from proteins produced inside the cell. Thus, class I MHC
complexes are useful for killing cells infected by viruses or cells
which have become cancerous as the result of expression of an
oncogene. T cells which have a protein called CD8 on their surface,
bind specifically to the MHC class I/peptide complexes via the T
cell receptor (TCR). This leads to cytolytic effector activities.
Class II MHC complexes are found only on antigen-presenting cells
(APC) and are used to present peptides from circulating pathogens
which have been endocytosed by APCs. T cells which have a protein
called CD4 bind to the MHC class II/peptide complexes via TCR. This
leads to the synthesis of specific cytokines which stimulate an
immune response. To be effectively recognized by the immune system
via MHC class I presentation, an antigenic polypeptide has to
contain an epitope of at least about 8 to 10 amino acids, while to
be effectively recognized by the immune system via MHC class II
presentation, an antigenic polypeptide has to contain an epitope of
at least about 13 to 25 amino acids. See, e.g., Fundamental
Immunology, 3.sup.rd Edition, W. E. Paul ed., 1999,
Lippincott-Raven Publ.
[0073] The term "species-specific" antigen refers to an antigen
that is only present in or derived from a particular species. Thus,
the term "malaria-derived" or "malaria-specific" antigen refers to
a natural (e.g., irradiated sporozoites) or synthetic (e.g.,
chemically produced multiple antigen peptide [MAP] or recombinantly
synthesized polypeptide) antigen comprising at least one epitope (B
cell and/or T cell) derived from any one of the proteins
constituting plasmodium (said plasmodium being without limitation
P. falciparum, P. vivax, P. malariae, P. ovale, P. reichenowi, P.
knowlesi, P. cynomolgi, P. brasilianum, P. yoelii, P. berghei, or
P. chabaudi) and comprising at least 5-10 amino acid residues. A
preferred plasmodial protein for antigen generation is
circumsporozoite (CS) protein, however, other proteins can be also
used, e.g., Thrombospondin Related Adhesion (Anonymous) protein
(TRAP), also called Sporozoite Surface Protein 2 (SSP2), LSA I,
hsp70, SALSA, STARP, Hep17, MSA, RAP-1, RAP-2, etc.
[0074] The term "vaccine" refers to a composition (e.g., protein or
vector such as, e.g., an adenoviral vector, Sindbis virus vector,
or pox virus vector) that can be used to elicit protective immunity
in a recipient. It should be noted that to be effective, a vaccine
of the invention can elicit immunity in a portion of the immunized
population, as some individuals may fail to mount a robust or
protective immune response, or, in some cases, any immune response.
This inability may stem from the individual's genetic background or
because of an immunodeficiency condition (either acquired or
congenital) or immunosuppression (e.g., due to treatment with
chemotherapy or use of immunosuppressive drugs, e.g., to prevent
organ rejection or suppress an autoimmune condition). Vaccine
efficacy can be established in animal models.
[0075] The term "DNA vaccine" is an informal term of art, and is
used herein to refer to a vaccine delivered by means of a
recombinant vector. An alternative, and more descriptive term used
herein is "vector vaccine" (since some potential vectors, such as
retroviruses and lentiviruses are RNA viruses, and since in some
instances non-viral RNA instead of DNA is delivered to cells
through the vector). Generally, the vector is administered in vivo,
but ex vivo transduction of appropriate antigen presenting cells,
such as dendritic cells (DC), with administration of the transduced
cells in vivo, is also contemplated.
[0076] The term "treat" is used herein to mean to relieve or
alleviate at least one symptom of a disease in a subject. Within
the meaning of the present invention, the term "treat" may also
mean to prolong the prepatency, i.e., the period between infection
and clinical manifestation of a disease. Preferably, the disease is
either infectious disease (e.g., viral, bacterial, parasitic, or
fungal) or malignancy (e.g., solid or blood tumors such as
sarcomas, carcinomas, gliomas, blastomas, pancreatic cancer, breast
cancer, ovarian cancer, prostate cancer, lymphoma, leukemia,
melanoma, etc.).
[0077] The term "protect" is used herein to mean prevent or treat,
or both, as appropriate, development or continuance of a disease in
a subject. Within the meaning of the present invention, the disease
is selected from the group consisting of infection (e.g., viral,
bacterial, parasitic, or fungal) and malignancy (e.g., solid or
blood tumors such as sarcomas, carcinomas, gliomas, blastomas,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
lymphoma, leukemia, melanoma, etc.). For example, as disclosed
herein, a prophylactic administration of an anti-malarial vaccine
comprising a plasmodium-derived antigen in combination with an
adjuvant comprising .alpha.-GalCer can protect a recipient subject
at risk of developing malaria. Similarly, according to the present
invention, a therapeutic administration of a tumor-specific antigen
conjointly with an adjuvant comprising a-GalCer or another
glycosylceramide of Formula 1 can enhance an anti-tumor immune
response leading to slow-down in tumor growth and metastasis or
even tumor regression.
[0078] The term "protective immunity" refers to an immune response
in a host animal (either active/acquired or passive/innate, or
both) which leads to inactivation and/or reduction in the load of
said antigen and to generation of long-lasting immunity (that is
acquired, e.g., through production of antibodies), which prevents
or delays the development of a disease upon repeated exposure to
the same or a related antigen. A "protective immune response"
comprises a humoral (antibody) immunity or cellular immunity, or
both, effective to, e.g., eliminate or reduce the load of a
pathogen or infected cell (or produce any other measurable
alleviation of the infection), or to reduce a tumor burden in an
immunized (vaccinated) subject. Within the meaning of the present
invention, protective immunity may be partial.
[0079] Immune systems are classified into two general systems, the
"innate" or "natural" immune system and the "acquired" or
"adaptive" immune system. It is thought that the innate immune
system initially keeps the infection under control, allowing time
for the adaptive immune system to develop an appropriate response.
Recent studies have suggested that the various components of the
innate immune system trigger and augment the components of the
adaptive immune system, including antigen-specific B and T
lymphocytes (Fearon and Locksley, supra; Kos, 1998, Immunol. Res.,
17: 303; Romagnani, 1992, Immunol. Today, 13: 379; Banchereau and
Steinman, 1988, Nature, 392: 245).
[0080] The term "innate immunity" or "natural immunity" refers to
innate immune responses that are not affected by prior contact with
the antigen. Cells of the innate immune system, including
macrophages and dendritic cells (DC), take up foreign antigens
through pattern recognition receptors, combine peptide fragments of
these antigens with MHC class I and class II molecules, and
stimulate naive CD8.sup.+ and CD4.sup.+ T cells respectively
(Banchereau and Steinman, supra; Holmskov et al., 1994, Immunol.
Today, 15: 67; Ulevitch and Tobias, 1995, Annu. Rev. Immunol., 13:
437). Professional antigen-presenting cells (APC) communicate with
these T cells leading to the differentiation of naive CD4.sup.+ T
cells into T-helper 1 (Th1) or T-helper 2 (Th2) lymphocytes that
mediate cellular and humoral immunity, respectively (Trinchieri,
1995, Annu. Rev. Immunol., 13: 251; Howard and O'Garra, 1992,
Immunol. Today, 13: 198; Abbas et al., 1996, Nature, 383: 787;
Okamura et al., 1998, Adv. Immunol., 70: 281; Mosmann and Sad,
1996, Immunol. Today, 17: 138; O'Garra, 1998, Immunity, 8:
275).
[0081] The term "acquired immunity" or "adaptive immunity" is used
herein to mean active or passive, humoral or cellular immunity that
is established during the life of an animal, is specific for the
inducing antigen, and is marked by an enhanced response on repeated
encounters with said antigen. A key feature of the T lymphocytes of
the adaptive immune system is their ability to detect minute
concentrations of pathogen-derived peptides presented by MHC
molecules on the cell surface.
[0082] As used herein, the term "augment the immune response" means
enhancing or extending the duration of the immune response, or
both. When referred to a property of an agent (e.g., adjuvant), the
term "[able to] augment the immunogenicity" refers to the ability
to enhance the immunogenicity of an antigen or the ability to
extend the duration of the immune response to an antigen, or
both.
[0083] The phrase "enhance immune response" within the meaning of
the present invention refers to the property or process of
increasing the scale and/or efficiency of immunoreactivity to a
given antigen, said immunoreactivity being either humoral or
cellular immunity, or both. An immune response is believed to be
enhanced, if any measurable parameter of antigen-specific
immunoreactivity (e.g., antibody titer, T cell production) is
increased at least two-fold, preferably ten-fold, most preferably
thirty-fold.
[0084] The term "therapeutically effective" applied to dose or
amount refers to that quantity of a compound or pharmaceutical
composition or vaccine that is sufficient to result in a desired
activity upon administration to a mammal in need thereof. As used
herein with respect to adjuvant--and antigen-containing
compositions or vaccines, the term "therapeutically effective
amount/dose" is used interchangeably with the term "immunogenically
effective amount/dose" and refers to the amount/dose of a compound
(e.g., an antigen and/or an adjuvant comprising glycosylceramide)
or pharmaceutical composition or vaccine that is sufficient to
produce an effective immune response upon administration to a
mammal.
[0085] The phrase "pharmaceutically acceptable", as used in
connection with compositions of the invention, refers to molecular
entities and other ingredients of such compositions that are
physiologically tolerable and do not typically produce untoward
reactions when administered to a human. Preferably, as used herein,
the term "pharmaceutically acceptable" means approved by a
regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in mammals, and more particularly in humans.
[0086] The term "carrier" applied to pharmaceutical or vaccine
compositions of the invention refers to a diluent, excipient, or
vehicle with which a compound (e.g., an antigen and/or an adjuvant
comprising glycosylceramide) is administered. Such pharmaceutical
carriers can be sterile liquids, such as water and oils, including
those of petroleum, animal, vegetable or synthetic origin, such as
peanut oil, soybean oil, mineral oil, sesame oil and the like.
Water or aqueous solution, saline solutions, and aqueous dextrose
and glycerol solutions are preferably employed as carriers,
particularly for injectable solutions. Suitable pharmaceutical
carriers are described in "Remington's Pharmaceutical Sciences" by
E. W. Martin, 18.sup.th Edition.
[0087] The term "native antibodies" or "immunoglobulins" refers to
usually heterotetrameric glycoproteins of about 150,000 daltons,
composed of two identical light (L) chains and two identical heavy
(H) chains. Each light chain is linked to a heavy chain by one
covalent disulfide bond, while the number of disulfide linkages
varies between the heavy chains of different immunoglobulin
isotypes. Each heavy and light chain also has regularly spaced
intrachain disulfide bridges. Each heavy chain has at one end a
variable domain (VH) followed by a number of constant domains. Each
light chain has a variable domain (VL) at one end and a constant
domain at its other end; the constant domain of the light chain is
aligned with the first constant domain of the heavy chain, and the
light chain variable domain is aligned with the variable domain of
the heavy chain. Particular amino acid residues are believed to
form an interface between the light and heavy chain variable
domains (Clothia et al., J Mol. Biol., 186: 651-663, 1985; Novotny
and Haber, Proc. Natl. Acad. Sci. USA, 82: 4592-4596, 1985).
[0088] The term "antibody" or "Ab" is used in the broadest sense
and specifically covers not only native antibodies but also single
monoclonal antibodies (including agonist and antagonist
antibodies), antibody compositions with polyepitopic specificity,
as well as antibody fragments (e.g., Fab, F(ab')2, scFv and Fv), so
long as they exhibit the desired biological activity.
[0089] "Cytokine" is a generic term for a group of proteins
released by one cell population which act on another cell
population as intercellular mediators. Examples of such cytokines
are lymphokines, monokines, and traditional polypeptide hormones.
Included among the cytokines are interferons (IFN, notably
IFN-.gamma.), interleukins (IL, notably IL-1, IL-2, IL-4, IL-10,
IL-12), colony stimulating factors (CSF), thrombopoietin (TPO),
erythropoietin (EPO), leukemia inhibitory factor (LIF), kit-ligand,
growth hormones (GH), insulin-like growth factors (IGF),
parathyroid hormone, thyroxine, insulin, relaxin, follicle
stimulating hormone (FSH), thyroid stimulating hormone (TSH),
leutinizing hormone (LH), hematopoietic growth factor, hepatic
growth factor, fibroblast growth factors (FGF), prolactin,
placental lactogen, tumor necrosis factors (TNF),
mullerian-inhibiting substance, mouse gonadotropin-associated
peptide, inhibin, activin, vascular endothelial growth factor
(VEGF), integrin, nerve growth factors (NGF), platelet growth
factor, transforming growth factors (TGF), osteoinductive factors,
etc.
[0090] The term "subject" as used herein refers to an animal having
an immune system, preferably a mammal (e.g., rodent such as mouse).
In particular, the term refers to humans.
[0091] The term "about" or "approximately" usually means within
20%, more preferably within 10%, and most preferably still within
5% of a given value or range. Alternatively, especially in
biological systems (e.g., when measuring an immune response), the
term "about" means within about a log (i.e., an order of magnitude)
preferably within a factor of two of a given value.
[0092] The terms "vector", "cloning vector", and "expression
vector" mean the vehicle by which a DNA or RNA sequence (e.g., a
foreign gene) can be introduced into a host cell, so as to
transform the host and promote expression (e.g., transcription
and/or translation) of the introduced sequence. Vectors include
plasmids, phages, viruses, etc.
[0093] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are well-known and are explained fully in the
literature. See, e.g., Sambrook, Fritsch and Maniatis, Molecular
Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York (herein
"Sambrook et al., 1989"); DNA Cloning: A Practical Approach,
Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis
(M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames
& S. J. Higgins eds. (1985)]; Transcription And Translation [B.
D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R.
I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press,
(1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984);
F. M. Ausubel et al. (eds.), Current Protocols in Molecular
Biology, John Wiley & Sons, Inc. (1994).
[0094] A "nucleic acid molecule" (or alternatively "nucleic acid")
refers to the phosphate ester polymeric form of ribonucleosides
(adenosine, guanosine, uridine, or cytidine: "RNA molecules") or
deoxyribonucleosides (deoxyadenosine, deoxyguanosine,
deoxythymidine, or deoxycytidine: "DNA molecules"), or any
phosphoester analogs thereof, such as phosphorothioates and
thioesters, in either single stranded form, or a double-stranded
helix. Oligonucleotides (having fewer than 100 nucleotide
constituent units) or polynucleotides are included within the
defined term as well as double stranded DNA-DNA, DNA-RNA, and
RNA-RNA helices. This term, for instance, includes double-stranded
DNA found, inter alia, in linear (e.g., restriction fragments) or
circular DNA molecules, plasmids, and chromosomes. In discussing
the structure of particular double-stranded DNA molecules,
sequences may be described herein according to the normal
convention of giving only the sequence in the 5' to 3' direction
along the nontranscribed strand of DNA (i.e., the strand having a
sequence homologous to the mRNA). A "recombinant DNA molecule" is a
DNA molecule that has undergone a molecular biological
manipulation.
[0095] As used herein, the term "polypeptide" refers to an amino
acid-based polymer, which can be encoded by a nucleic acid or
prepared synthetically. Polypeptides can be proteins, protein
fragments, chimeric proteins, etc. Generally, the term "protein"
refers to a polypeptide expressed endogenously in a cell.
Generally, a DNA sequence encoding a particular protein or enzyme
is "transcribed" into a corresponding sequence of mRNA. The mRNA
sequence is, in turn, "translated" into the sequence of amino acids
which form a protein. An "amino acid sequence" is any chain of two
or more amino acids. The term "peptide" is usually used for amino
acid-based polymers having fewer than 100 amino acid constituent
units, whereas the term "polypeptide" is reserved for polymers
having at least 100 such units. Herein, however, "polypeptide" will
be the generic term.
[0096] Uses of Adjuvants Comprising Glycosylceramides
[0097] In one aspect, the present invention provides a method for
augmenting an immunogenicity of an antigen in a mammal, comprising
administering said antigen conjointly with an adjuvant composition
comprising a glycosylceramide of Formula 1, preferably
.alpha.-galactosyl-ceramide (.alpha.-GalCer). According to the
present invention, the use of glycosylceramide as an adjuvant
results in an enhancement and/or extension of the duration of the
protective immunity induced by the antigen. For example, as
disclosed herein, conjoint administration of glycosylceramide with
peptides corresponding to T cell or B cell epitopes of tumor or
viral antigens, or DNA constructs expressing these antigens
enhances antigen-specific immune responses.
[0098] The glycosylceramide-containing adjuvant of the invention
can be conjointly administered with any antigen, in particular,
with antigens derived from infectious agents or tumors.
[0099] As discussed in the Background Section, the
immunostimulating effects of glycosylceramides both in mice and
humans depend on the expression of CD1d molecules and are mediated
by NKT cells. Indeed, the instant invention demonstrates that
.alpha.-GalCer adjuvant activity is attributed at least in part to
its ability to enhance and/or extend NKT-mediated antigen-specific
Th1-type T cell responses and CD8+ T cell (or Tc) responses.
[0100] From an immunotherapy view point, glycosylceramide-mediated
activation of the NKT cell system appears to have distinct
advantages over the other mechanisms for the following reasons: (a)
the level of cytotoxicity of activated NKT cells is very high and
effective against a wide variety of tumor cells or infected cells;
(b) the activation of NKT cells by glycosylceramide is totally
dependent on a CD1d molecule, which is monomorphic among
individuals (Porcelli, Adv. Immunol., 59: 1-98, 1995), indicating
that glycosylceramide-containing adjuvants can be utilized by all
patients, regardless of MHC haplotype; (c) antigen-presenting
functions of DC and NKT activation of human patients can be
evaluated before immunotherapy by the in vivo assays in mice using
V.alpha.14 NKT cell status as an indicator.
[0101] According to the present invention, an adjuvant comprising
glycosylceramide of Formula 1 and antigen can be administered
either as two separate formulations or as part of the same
composition. If administered separately, the adjuvant and antigen
can be administered either sequentially or simultaneously. As
disclosed herein, simultaneous administration of glycosylceramide
adjuvant with the antigen is preferred and generally allows to
achieve the most efficient immunostimulation.
[0102] As the glycosylceramide adjuvant of the invention exerts its
immunostimulatory activity in combination with a plurality of
different antigens, it is therefore useful for both preventive and
therapeutic applications. Accordingly, in a further aspect, the
invention provides a prophylactic and/or therapeutic method for
treating a disease in a mammal comprising conjointly administering
to said mammal an antigen and an adjuvant comprising a
glycosyl-ceramide of Formula 1. This method can be useful, e.g.,
for protecting against and/or treating various infections as well
as for treating various neoplastic diseases.
[0103] Immunogenicity enhancing methods of the invention can be
used to combat infections, which include, but are not limited to,
parasitic infections (such as those caused by plasmodial species,
etc.), viral infections (such as those caused by influenza viruses,
leukemia viruses, immunodeficiency viruses such as HIV, papilloma
viruses, herpes virus, hepatitis viruses, measles virus,
poxviruses, mumps virus, cytomegalovirus [CMV], Epstein-Barr virus,
etc.), bacterial infections (such as those caused by
staphylococcus, streptococcus, pneumococcus, Neisseria gonorrhea,
Borrelia, pseudomonas, etc.), and fungal infections (such as those
caused by candida, trichophyton, ptyrosporum, etc.).
[0104] Methods of the invention are also useful in treatment of
various cancers, which include without limitation fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendothelio-sarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
lymphoma, leukemia, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma, and
retinoblastoma.
[0105] As further disclosed herein, maximal efficiency of the
immunogenicity enhancing methods of present invention is attained
when an antigen and glycosylceramide adjuvant are administered
simultaneously.
[0106] In a specific embodiment, the present invention discloses a
method for preventing and/or treating malaria in a mammal (e.g.,
human), wherein said method comprises conjointly administering to
said mammal a malaria-specific antigen and an adjuvant comprising a
glycosylceramide of Formula 1, preferably, .alpha.-GalCer. As
disclosed in Example 1, infra, the immunization of mice with a
sub-optimal dose of irradiated malaria parasites, co-administered
with .alpha.-GalCer, greatly enhances protective anti-malaria
immunity. The .alpha.-GalCer co-administration not only increases
the level of protection but also prolongs the duration of
protective anti-malaria immunity. Furthermore, it is disclosed
herein that co-injection of mice with .alpha.-GalCer and irradiated
parasites or peptides (corresponding to CD4+ or CD8+ epitopes of
the malarial CS protein), leads to an increase in the number of
antigen-specific T cells.
[0107] In another specific embodiment, the invention discloses a
method for enhancing the immune response to HIV infection (and
potentially preventing and/or treating AIDS) in a mammal, wherein
said method comprises conjointly administering to said mammal an
HIV-specific antigen and an .alpha.-GalCer adjuvant. As disclosed
in Example 2, infra, co-administration of .alpha.-GalCer to mice
immunized with a CD8+ T cell epitope (RGPGRAFVTI [SEQ ID NO: 5]) of
p18 (V3 loop) of HIV, enhances almost 3 times the level of
HIV-specific CD8+ T cell response compared to that induced in mice
immunized without .alpha.-GalCer treatment.
[0108] The methods of the invention can be used in conjunction with
other treatments. For example, an anti-cancer treatment using
tumor-specific antigen and glycosylceramide-containing adjuvant of
the present invention can be used in combination with chemotherapy
and/or radiotherapy and/or IL-12 treatment. Anti-viral vaccines
comprising .alpha.-glycosyl-ceramide- -containing adjuvant can be
used in combination with IFN-.alpha.treatment.
[0109] In addition to its therapeutic applications, the
glycosylceramide adjuvant of the invention may be also applied as a
research tool to the study of many aspects of basic immunology. For
example, it can be used to study immune mechanisms, such as
function of NKT cells, antigen presentation by DC, and modulation
of immune responses by cytokines and their receptors.
Glycosylceramide adjuvant can be also employed in vaccine design
research, which could assist in identifying the requirements for
protective immunity, since for the same antigen different adjuvants
may produce immune responses of varying intensity and/or
length.
[0110] Glycosylceramide-Containing Pharmaceutical and Vaccine
Compositions
[0111] In conjunction with the methods of the present invention,
also provided are pharmaceutical and vaccine compositions
comprising an immunogenically effective amount of an antigen and
immunogenically effective amount of an adjuvant comprising
glycosylceramide as well as, optionally, an additional
immunostimulant, carrier or excipient (preferably all
pharmaceutically acceptable). Said antigen and adjuvant can be
either formulated as a single composition or as two separate
compositions, which can be administered simultaneously or
sequentially.
[0112] Adjuvants of the invention comprise compounds which belong
to the class of sphingoglycolipids, specifically glycosylceramides,
which can be represented by a general Formula 1: 10
[0113] wherein R.sub.1, R.sub.2 and R.sub.5 represent H or a
specific monosaccharide; R.sub.3 and R.sub.6 represent H or OH,
respectively; R.sub.4 represents H, OH or a specific
monosaccharide; X denotes an integer from 1 to 23; R.sub.7
represents any one of the following groups (a)-(g): (a)
--(CH.sub.2).sub.11--CH.sub.3, (b) --(CH.sub.2).sub.12--CH.s- ub.3,
(c) --(CH.sub.2).sub.13--CH.sub.3, (d)
--(CH.sub.2).sub.9--CH(CH.sub- .3).sub.2, (e)
--(CH.sub.2).sub.10--CH(CH.sub.3).sub.2, (f)
--(CH.sub.2).sub.11--CH(CH.sub.3).sub.2, (g)
--(CH.sub.2).sub.11--CH(CH.s- ub.3)--C.sub.2H.sub.5.
[0114] A preferred adjuvant of the present invention comprises
a-galactosylceramide (.alpha.-GalCer), specifically, (2S,3S,4R)-
1-O-(.alpha.-D-galactopyranosyl)-2-(N-hexacosanoylamino)-
1,3,4-octadecanetriol represented by the Formula 2: 11
[0115] Other examples of glycosylceramides useful in adjuvants of
the present invention include, without limitation:
[0116] .alpha.-glucosylceramide (.alpha.-GlcCer), specifically
(2S,3S,4R)-1-O-(.alpha.-D-glucopyranosyl)-N-hexacosanoyl-2-amino-1,3,4-oc-
tadecanetriol, of the Formula 3: 12
[0117] Gal.alpha.1-6Gal.alpha.1-1'Cer, specifically
(2S,3S,4R)-2-amino-
1-O-(.alpha.-D-galactopyranosyl-(1-6)-.alpha.-D-galactopyranosyl)-N-hexac-
osanoyl-1,3,4-octadecanetriol, of the Formula 4: 13
[0118] Gal.alpha.1-6Glc.alpha.1-1'Cer, specifically
(2S,3S,4R)-2-amino-1-O-(.alpha.-D-galactopyranosyl-(1-6)-.alpha.-D-
glucopyranosyl)-N-hexacosanoyl- 1,3,4-octadecanetriol, of the
Formula 5: 14
[0119] Gal.alpha.1-2Glc.alpha.1-1'Cer, specifically
(2S,3S,4R)-2-amino-1-O-(.alpha.-D-glucopyranosyl-(1-2)-.alpha.-D-galactop-
yranosyl)-N-[(R)-2-hydroxytetracosanoyl]-1,3,4-octadecanetriol, of
the Formula 6: 15
[0120] Gal.beta.1-3Gal.alpha.1-1'Cer, specifically
(2S,3S,4R)-2-amino-1-O--
(.beta.-D-galactofuranosyl-(1-4)-.alpha.-D-galactopyranosyl)-N-[(R)-2-hydr-
oxytetracosanoyl]-1,3,4-octadecanetriol, of the Formula 7: 16
[0121] .alpha.-GalCer adjuvant component can be either isolated
from Okinawan marine sponges (e.g., as described by Natori et al.,
Tetrahedron, 50: 2771-2784, 1994) or produced synthetically (see,
e.g., U.S. Pat. Nos. 5,936,076; 5,780,441; 5,849,716, and
6,071,884; PCT Publication Nos. WO 98/29534, and WO 98/44928;
Kobayashi et al., 1995, Oncol. Res., 7:529-534). Similarly, other
related glycosylceramide adjuvants of the invention can be either
isolated from a natural source (e.g., marine sponges) or produced
synthetically (as described in, e.g., U.S. Pat. Nos. 5,936,076;
5,780,441; 5,849,716, and 6,071,884; PCT Publication Nos. WO
98/29534 and WO 98/44928; Morita et al., J. Med. Chem.,
38:2176-2187, 1995; Teriyuki et al., J. Med. Chem., 42:1836-1841,
1999).
[0122] The antigens used in immunogenic (e.g., vaccine)
compositions of the instant invention can be derived from a
eukaryotic cell (e.g., tumor, parasite, fungus), bacterial cell,
viral particle, or any portion thereof. In the event the material
to which the immunogenic response is to be directed is poorly
antigenic, it may be additionally conjugated to a carrier molecule
such as albumin or hapten, using standard covalent binding
techniques, for example, with one of the several commercially
available reagent kits.
[0123] Examples of preferred antigens of the present invention
include (i) malaria-specific antigens such as irradiated plasmodial
sporozoites or synthetic peptide antigens comprising at least one T
cell and/or B cell epitope of the malarial circumsporozoite (CS)
protein (see below); (ii) viral protein or peptide antigens such as
those derived from influenza virus (e.g., surface glycoproteins
hemagluttinin (HA) and neuraminidase (NA) [such as turkey influenza
HA or an avian influenza A/Jalisco/95 H5 HA); immunodeficiency
virus (e.g., a feline immunodeficiency virus (FIV) antigen, a
simian immunodeficiency virus (SIV) antigen, or a human
immunodeficiency virus antigen (HIV) such as gp120, gp160, p18
antigen [described in Example 2, infra]), Gag p17/p24, Tat, Pol,
Nef, and Env; herpesvirus (e.g., a glycoprotein, for instance, from
feline herpesvirus, equine herpesvirus, bovine herpesvirus,
pseudorabies virus, canine herpesvirus, herpes simplex virus (HSV,
e.g., HSV tk, gB, gD), Marek's Disease Virus, herpesvirus of
turkeys (HVT), or cytomegalovirus (CMV), or Epstein-Barr virus);
hepatitis virus (e.g., Hepatitis B surface antigen (HBsAg));
papilloma virus; bovine leukemia virus (e.g., gp51,30 envelope
antigen); feline leukemia virus (FeLV) (e.g., FeLV envelope
protein, a Newcastle Disease Virus (NDV) antigen, e.g., HN or F);
rous associated virus (such as RAV-1 env); infectious bronchitis
virus (e.g., matrix and/or preplomer); flavivirus (e.g., a Japanese
encephalitis virus (JEV) antigen, a Yellow Fever antigen, or a
Dengue virus antigen); Morbillivirus (e.g., a canine distemper
virus antigen, a measles antigen, or rinderpest antigen such as HA
or F); rabies (e.g., rabies glycoprotein G); parvovirus (e.g., a
canine parvovirus antigen); poxvirus (e.g., an ectromelia antigen,
a canary poxvirus antigen, or a fowl poxvirus antigen); chicken pox
virus (varicella zoster antigen); infectious bursal disease virus
(e.g., VP2, VP3, or VP4); Hantaan virus; mumps virus; (iii)
bacterial antigens such as lipopolysaccharides isolated from
gram-negative bacterial cell walls and staphylococcus-specific,
streptococcus-specific, pneumococcus-specific (e.g., PspA [see PCT
Publication No. WO 92/14488]), Neisseria gonorrhea-specific
Borrelia-specific (e.g., OspA, OspB, OspC antigens of Borrelia
associated with Lyme disease such as Borrelia burgdorferi, Borrelia
afzelli, and Borrelia garinii [see, e.g., U.S. Pat. No. 5,523,089;
PCT Publication Nos. WO 90/04411, WO 91/09870, WO 93/04175, WO
96/06165, W093/08306; PCT/US92/08697; Bergstrom et al., Mol.
Microbiol., 3: 479-486, 1989; Johnson et al., Infect. and Immun.
60: 1845-1853, 1992; Johnson et al., Vaccine 13: 1086-1094, 1995;
The Sixth International Conference on Lyme Borreliosis: Progress on
the Development of Lyme Disease Vaccine, Vaccine 13: 133-135,
1995]), and pseudomonas-specific proteins or peptides; (iv) fungal
antigens such as those isolated from candida, trichophyton, or
ptyrosporum, and (v) tumor-specific proteins such as ErbB
receptors, Melan A [MART1], gp100, tyrosinase, TRP-1/gp75, and
TRP-2 (in melanoma); MAGE-1 and MAGE-3 (in bladder, head and neck,
and non-small cell carcinoma); HPV EG and E7 proteins (in cervical
cancer); Mucin [MUC-1] (in breast, pancreas, colon, and prostate
cancers); prostate-specific antigen [PSA] (in prostate cancer);
carcinoembryonic antigen [CEA] (in colon, breast, and
gastrointestinal cancers) and such shared tumor-specific antigens
as MAGE-2, MAGE-4, MAGE-6, MAGE-10, MAGE-12, BAGE-1, CAGE-1,2,8,
CAGE-3 to 7, LAGE-1, NY-ESO-1/LAGE-2, NA-88, GnTV, and
TRP2-INT2.
[0124] The foregoing list of antigens are intended as exemplary, as
the antigen of interest can be derived from any animal or human
pathogen or tumor. With respect to DNA encoding pathogen-derived
antigens of interest, attention is directed to, e.g., U.S. Pat.
Nos. 4,722,848; 5,174,993; 5,338,683; 5,494,807; 5,503,834;
5,505,941; 5,514,375; 5,529,780; U.K. Patent No. GB 2 269 820 B;
and PCT Publication Nos. WO 92/22641; WO 93/03145; WO 94/16716; WO
96/3941; PCT/US94/06652. With respect to antigens derived from
tumor viruses, reference is also made to Molecular Biology of Tumor
Viruses, RNA Tumor Viruses, Second Edition, Edited by Weiss et al.,
Cold Spring Harbor Laboratory Press, 1982. For a list of additional
antigens useful in the compositions of the invention see also
Stedman's Medical Dictionary (24th edition, 1982).
[0125] In a specific embodiment, the compositions of the present
invention provide protective immunity against malaria, in
particular against P. yoelii and major human plasmodial species P.
falciparum and P. vivax. These compositions comprise one or more of
the following components: (i) at least one malaria-specific peptide
comprising a T cell epitope capable of eliciting an anti-malarial
T-cell response preferably in mammals of diverse genetic
backgrounds (e.g., YNRNIVNRLLGDALNGKPEEK [SEQ ID NO: 1] or
SYVPSAEQI [SEQ ID NO: 2] T cell epitope of P. yoelii CS protein
[Renia et al., J. Immunol., 22: 157-160, 1993; Rodrigues et al.,
Int. Immunol., 3: 579-585, 1991] or (NVDPNANP).sub.n [SEQ ID NO: 3]
or EYLNKIQNSLSTE WSPCSVT [SEQ ID NO: 4] T cell epitope of P.
falciparum CS protein [Nardin et al., Science 246:1603, 1989;
Moreno et al., Int.Immunol. 3: 997, 1991; Moreno et al., J.
Immunol. 151: 489, 1993]); (ii) at least one malaria-specific
peptide comprising a B cell epitope (e.g., (NANP).sub.3 [SEQ ID NO:
15] B cell epitope located within the repeat region of the CS
protein of P. falciparum [Nardin et al., J.Exp.Med. 156: 20, 1982;
Nardin et al., Ann. Rev. Immunol. 11: 687, 1993]) capable of
stimulating the production of anti-malarial (i.e., neutralizing)
antibodies (e.g., directed against the sporozoite stage of the
malarial organism). Preferably, the immunogenic compositions of the
present invention comprise at least one B cell epitope and at least
one T cell epitope. B cell epitopes preferably elicit the
production of antibodies that specifically recognize and bind to
the malarial circumsporozoite (CS) protein. Alternatively or in
addition, the compositions of the invention may comprise B cell
and/or T cell epitopes derived from, and reactive with, other
malarial components, such as, for example, the P. vivax Erythrocyte
Secreted Protein-1 or -2 (PvESP-1 or PvESP-2) (see, e.g., U.S. Pat.
No. 5,874,527), P. falciparum sporozoite surface protein designated
Thrombospondin Related Adhesion (Anonymous) protein (TRAP), also
called Sporozoite Surface Protein 2 (SSP2), LSA I, hsp70, SALSA,
STARP, Hep17, MSA, RAP-1, and RAP-2. In one embodiment, the B cell
epitope and T cell epitope components are incorporated into
multiple antigen peptides (MAPs), forming a synthetic
macromolecular polypeptide containing a high density of the
epitopes. Methods for MAP synthesis are well known in the art (see,
e.g., Tam, Proc. Natl. Acad. Sci. USA, 85: 5409, 1988; Tam, Meth.
Enzymol., 168: 7, 1989).
[0126] The present invention also encompasses B cell and T cell
epitopes derived from other plasmodial species, including without
limitation P. malariae, P. ovale, P. reichenowi, P. knowlesi, P.
cynomolgi, P. brasilianum, P. berghei, and P. chabaudi. These
epitopes typically comprise between 8 and 18 amino acid residues,
derived from a plasmodial protein.
[0127] In another specific embodiment, a preferred antigen of the
invention is HIV-specific (such as T cell epitope RGPGRAFVTI [SEQ
ID NO: 5] of p18 protein, see Example 2, infra). As disclosed
herein, compositions comprising such HIV-specific antigen(s) and an
adjuvant comprising glycosylceramide of Formula 1, preferably
.alpha.-GalCer, are capable of enhancing a T cell response to an
HIV antigen in a susceptible mammalian host.
[0128] In yet another specific embodiment, an antigen of the
invention is influenza A virus-specific. As disclosed herein,
co-administation of .alpha.-GalCer with a suboptimal dose (10.sup.5
p.f.u.) of a recombinant Sindbis virus expressing a CD8+T cell
epitope TYQRTRALV (SEQ ID NO: 16) of the nucleoprotein (NP) of the
influenza A virus (Tsuji et al., J. Virol., 72:6907-6910, 1998)
significantly enhances the CD8+T cell anti-influenza response in a
susceptible mammalian host.
[0129] To provide additional antigen-derived B and T cell epitopes
for use in the compositions of the present invention, these
epitopes may be identified by one or a combination of several
methods well known in the art, such as, for example, by (i)
fragmenting the antigen of interest into overlapping peptides using
proteolytic enzymes, followed by testing the ability of individual
peptides to bind to an antibody elicited by the full-length antigen
or to induce T cell or B cell activation (see, e.g., Janis Kuby,
Immunology, pp. 79-80, W. H. Freeman, 1992); (ii) preparing
synthetic peptides whose sequences are segments or analogs of a
given antigen (see, e.g., Alexander et al., 1994, Immunity,
1:751-61; Hammer et al., 1994, J. Exp. Med., 180:2353-8), or
constructs based on such segments, or analogs linked or fused to a
carrier or a heterologous antigen and testing the ability of such
synthetic peptides to elicit antigen-specific antibodies or T cell
activation (e.g., testing their ability to interact with MHC class
II molecules both in vitro and in vivo [see, e.g., O'Sullivan et
al., 1991, J. Immunol., 147:2663-9; Hill et al., 1991, J. Immunol.,
147:189-197]); for determination of T cell epitopes, peptides
should be at least 8 to 10 amino acids long to occupy the groove of
the MHC class I molecule and at least 13 to 25 amino acids long to
occupy the groove of MHC class II molecule, preferably, the
peptides should be longer; these peptides should also contain an
appropriate anchor motif which will enable them to bind to various
class I or class II MHC molecules with high enough affinity and
specificity to generate an immune response (see Bocchia et al.,
Blood 85: 2680-2684, 1995; Englehard, Ann. Rev. Immunol.12: 181,
1994); (iii) sequencing peptides associated with purified MHC
molecules (see, e.g., Nelson et al., 1997, PNAS, 94:628-33); (iv)
screening a peptide display library for high-affinity binding to
MHC class II molecules, TCR, antibodies raised against a
full-length antigen, etc. (see, e.g., Hammer et al., 1992, J. Exp.
Med., 176:1007-13); (v) computationally analyzing different protein
sequences to identify, e.g., hydrophilic stretches (hydrophilic
amino acid residues are often located on the surface of the protein
and are therefore accessible to the antibodies) and/or
high-affinity TCR or MHC class II allele-specific motifs, e.g., by
comparing the sequence of the protein of interest with published
structures of peptides associated with the MHC molecules (Mallios,
Bioinformatics, 15:432-439, 1999; Milik et al., Nat. Biotechnol.,
16:753-756, 1998; Brusic et al., Nuc. Acids Res, 26:368-371, 1998;
Feller and de la Cruz, Nature, 349:720-721, 1991); (vi) performing
an X-ray crystallographic analysis of the native antigen-antibody
complex (Janis Kuby, Immunology, p.80, W. H. Freeman, 1992), and
(vii) generating monoclonal antibodies to various portions of the
antigen of interest, and then ascertaining whether those antibodies
attenuate in vitro or in vivo growth of the pathogen or tumor from
which the antigen was derived (see U.S. Pat. No. 5,019,384 and
references cited therein).
[0130] In a specific embodiment, the antigen of the invention may
be presented by a recombinant virus expressing said antigen.
Preferably, the virus is selected from the group consisting of a
recombinant adenovirus, recombinant pox virus, and recombinant
Sindbis virus.
[0131] In the disclosed compositions, both the antigen and the
glycosylceramide adjuvant are present in immunogenically effective
amounts. For each specific antigen, the optimal immunogenically
effective amount should be determined experimentally (taking into
consideration specific characteristics of a given patient and/or
type of treatment). Generally, this amount is in the range of 0.1
.mu.g-100 mg of an antigen per kg of the body weight. For the
glycosylceramide adjuvant of the present invention, the optimal
immunogenically effective amount is preferably in the range of
10-100 .mu.g of the adjuvant per kg of the body weight.
[0132] The invention also provides a method for preparing a vaccine
composition comprising at least one antigen and an adjuvant
comprising glycosylceramide of Formula 1, preferably
.alpha.-GalCer, said method comprising admixing the adjuvant and
the antigen, and optionally one or more physiologically acceptable
carriers and/or excipients and/or auxiliary substances.
[0133] Formulations and Administration
[0134] The invention provides pharmaceutical and vaccine
formulations containing therapeutics of the invention (an antigen
and glycosylceramide adjuvant either as a single composition or as
two separate compositions which can be administered simultaneously
or sequentially), which formulations are suitable for
administration to elicit an antigen-specific protective immune
response for the treatment and prevention of infectious or
neoplastic diseases described above. Compositions of the present
invention can be formulated in any conventional manner using one or
more physiologically acceptable carriers or excipients. Thus, an
antigen and/or an adjuvant comprising a glycosylceramide of Formula
1, preferably .alpha.-GalCer, can be formulated for administration
by transdermal delivery, or by transmucosal administration,
including but not limited to, oral, buccal, intranasal, opthalmic,
vaginal, rectal, intracerebral, intradermal, intramuscular,
intraperitoneal, intravenous, subcutaneous routes, via
scarification (scratching through the top layers of skin, e.g.,
using a bifurcated needle), by inhalation (pulmonary) or
insufflation (either through the mouth or the nose), or by
administration to antigen presenting cells ex vivo followed by
administration of the cells to the subject, or by any other
standard route of immunization.
[0135] Preferably, the immunogenic formulations of the invention
can be delivered parenterally, i.e., by intravenous (i.v.),
subcutaneous (s.c.), intraperitoneal (i.p.), intramuscular (i.m.),
subdermal (s.d.), or intradermal (i.d.) administration, by direct
injection, via, for example, bolus injection, continuous infusion,
or gene gun (e.g., to administer a vector vaccine to a subject,
such as naked DNA or RNA). Formulations for injection can be
presented in unit dosage form, e.g., in ampoules or in multi-dose
containers, with an added preservative. The compositions can take
such forms as excipients, suspensions, solutions or emulsions in
oily or aqueous vehicles, and can contain formulatory agents such
as suspending, stabilizing and/or dispersing agents. Alternatively,
the active ingredient can be in powder form for reconstitution with
a suitable vehicle, e.g., sterile pyrogen-free water, before
use.
[0136] The present invention also contemplates various mucosal
vaccination strategies. While the mucosa can be targeted by local
delivery of a vaccine, various strategies have been employed to
deliver immunogenic compositions to the mucosa. For example, in a
specific embodiment, the immunogenic polypeptide or vector vaccine
can be administered in an admixture with, or as a conjugate or
chimeric fusion protein with, cholera toxin, such as cholera toxin
B or a cholera toxin A/B chimera (see, e.g., Hajishengallis, J
Immunol., 154: 4322-32, 1995; Jobling and Holmes, Infect Immun.,
60: 4915-24, 1992; Lebens and Holmgren, Dev Biol Stand 82:215-27,
1994). In another embodiment, an admixture with heat labile
enterotoxin (LT) can be prepared for mucosal vaccination. Other
mucosal immunization strategies include encapsulating the immunogen
in microcapsules (see, e.g., U.S. Pat. Nos. 5,075,109; 5,820,883,
and 5,853,763) and using an immunopotentiating membranous carrier
(see, e.g., PCT Application No. WO 98/0558). Immunogenicity of
orally administered immunogens can be enhanced by using red blood
cells (rbc) or rbc ghosts (see, e.g., U.S. Pat. No. 5,643,577), or
by using blue tongue antigen (see, e.g., U.S. Pat. No. 5,690,938).
Systemic administration of a targeted immunogen can also produce
mucosal immunization (see, U.S. Pat. No. 5,518,725). Various
strategies can be also used to deliver genes for expression in
mucosal tissues, such as using chimeric rhinoviruses (see, e.g.,
U.S. Pat. No. 5,714,374), adenoviruses, vaccinia viruses, or
specific targeting of a nucleic acid (see, e.g., PCT Application
No. WO 97/05267).
[0137] For oral administration, the formulations of the invention
can take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinized maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets can be
coated by methods well known in the art. The compositions of the
invention can be also introduced in microspheres or microcapsules,
e.g., fabricated from poly-glycolic acid/lactic acid (PGLA) (see,
U.S. Pat. Nos. 5,814,344; 5,100,669 and 4,849,222; PCT Publication
Nos. WO 95/11010 and WO 93/07861). Liquid preparations for oral
administration can take the form of, for example, solutions,
syrups, emulsions or suspensions, or they can be presented as a dry
product for reconstitution with water or other suitable vehicle
before use. Such liquid preparations can be prepared by
conventional means with pharmaceutically acceptable additives such
as suspending agents (e.g., sorbitol syrup, cellulose derivatives
or hydrogenated edible fats); emulsifying agents (e.g., lecithin or
acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl
alcohol or fractionated vegetable oils); and preservatives (e.g.,
methyl or propyl-p-hydroxybenzoates or sorbic acid). The
preparations can also contain buffer salts, flavoring, coloring and
sweetening agents as appropriate. Preparations for oral
administration can be suitably formulated to give controlled
release of the active compound.
[0138] For administration by inhalation, the therapeutics according
to the present invention can be conveniently delivered in the form
of an aerosol spray presentation from pressurized packs or a
nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoro-methane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit can be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of, e.g., gelatin for use in an inhaler or insufflator
can be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0139] Compositions of the present invention can also be formulated
in rectal compositions such as suppositories or retention enemas,
e.g., containing conventional suppository bases such as cocoa
butter or other glycerides.
[0140] In addition to the formulations described previously, the
compositions can also be formulated as a depot preparation. Such
long acting formulations can be administered by implantation (for
example, subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds can be formulated with
suitable polymeric or hydrophobic materials (for example, as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0141] As disclosed herein, an antigen and/or glycosylceramide
adjuvant can be mixed with excipients which are pharmaceutically
acceptable and compatible with the active ingredients. Suitable
excipients are, for example, water, saline, buffered saline,
dextrose, glycerol, ethanol, sterile isotonic aqueous buffer or the
like and combinations thereof. In addition, if desired, the
preparations may also include minor amounts of auxiliary substances
such as wetting or emulsifying agents, pH buffering agents, and/or
immune stimulators (e.g., adjuvants in addition to
glycosylceramide) that enhance the effectiveness of the
pharmaceutical composition or vaccine. Non-limiting examples of
additional immune stimulators which may enhance the effectiveness
of the compositions of the present invention include
immunostimulatory, immunopotentiating, or pro-inflammatory
cytokines, lymphokines, or chemokines or nucleic acids encoding
them (specific examples include interleukin (IL)-1, IL-2, IL-3,
IL-4, IL-12, IL-13, granulocyte-macrophage (GM)-colony stimulating
factor (CSF) and other colony stimulating factors, macrophage
inflammatory factor, Flt3 ligand, see additional examples of
immunostimulatory cytokines in the Section entitled "Definitions").
These additional immunostimulatory molecules can be delivered
systemically or locally as proteins or by expression of a vector
that codes for expression of the molecule. The techniques described
above for delivery of the antigen and glycosylceramide adjuvant can
also be employed for the delivery of additional immunostimulatory
molecules.
[0142] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the immunogenic formulations of the invention. In a
related embodiment, the present invention provides a kit for the
preparation of a pharmaceutical or vaccine composition comprising
at least one antigen and a glycosylceramide-containing adjuvant,
said kit comprising the antigen in a first container, and the
adjuvant in a second container, and optionally instructions for
admixing the antigen and the adjuvant and/or for administration of
the composition. Each container of the kit may also optionally
include one or more physiologically acceptable carriers and/or
excipients and/or auxiliary substances. Associated with such
container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration.
[0143] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient (i.e., an antigen and/or a
glycosylceramide-containing adjuvant). The pack may, for example,
comprise metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. Compositions of the invention formulated in a
compatible pharmaceutical carrier may also be prepared, placed in
an appropriate container, and labeled for treatment of an indicated
condition.
[0144] Effective Dose and Safety Evaluations
[0145] According to the methods of the present invention, the
pharmaceutical and vaccine compositions described herein are
administered to a patient at immunogenically effective doses,
preferably, with minimal toxicity. As recited in the Section
entitled "Definitions", "immunogenically effective dose" or
"therapeutically effective dose" of disclosed formulations refers
to that amount of an antigen and/or glycosylceramide adjuvant that
is sufficient to produce an effective immune response in the
treated subject and therefore sufficient to result in a healthful
benefit to said subject.
[0146] Following methodologies which are well-established in the
art (see, e.g., reports on evaluation of several vaccine
formulations containing novel adjuvants in a collaborative effort
between the Center for Biological Evaluation and Food and Drug
Administration and the National Institute of Allergy and Infectious
Diseases [Goldenthal et al., National Cooperative Vaccine
Development Working Group. AIDS Res. Hum. Retroviruses, 1993,
9:545-9]), effective doses and toxicity of the compounds and
compositions of the instant invention are first determined in
preclinical studies using small animal models (e.g., mice) in which
both the antigen and glycosylceramide-containing adjuvant has been
found to be immunogenic and that can be reproducibly immunized by
the same route proposed for the human clinical trials.
Specifically, for any pharmaceutical composition or vaccine used in
the methods of the invention, the therapeutically effective dose
can be estimated initially from animal models to achieve a
circulating plasma concentration range that includes the IC.sub.50
(i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms). Dose-response curves derived
from animal systems are then used to determine testing doses for
the initial clinical studies in humans. In safety determinations
for each composition, the dose and frequency of immunization should
meet or exceed those anticipated for use in the clinical trial.
[0147] As disclosed herein, the dose of glycosylceramide,
antigen(s) and other components in the compositions of the present
invention is determined to ensure that the dose administered
continuously or intermittently will not exceed a certain amount in
consideration of the results in test animals and the individual
conditions of a patient. A specific dose naturally varies depending
on the dosage procedure, the conditions of a patient or a subject
animal such as age, body weight, sex, sensitivity, feed, dosage
period, drugs used in combination, seriousness of the disease. The
appropriate dose and dosage times under certain conditions can be
determined by the test based on the above-described indices and
should be decided according to the judgment of the practitioner and
each patient's circumstances according to standard clinical
techniques. In this connection, the dose of an antigen is generally
in the range of 0.1 .mu.g-100 mg per kg of the body weight, and the
dose of the glycosylceramide adjuvant required for augmenting the
immune response to the antigen is generally in the range of 10-100
.mu.g per kg of the body weight.
[0148] Toxicity and therapeutic efficacy of
glycosylceramide-containing immunogenic compositions of the
invention can be determined by standard pharmaceutical procedures
in experimental animals, e.g., by determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.5/ED.sub.50. Compositions
that exhibit large therapeutic indices are preferred. While
therapeutics that exhibit toxic side effects can be used (e.g.,
when treating severe forms of cancer or life-threatening
infections), care should be taken to design a delivery system that
targets such immunogenic compositions to the specific site (e.g.,
lymphoid tissue mediating an immune response, tumor or an organ
supporting replication of the infectious agent) in order to
minimize potential damage to other tissues and organs and, thereby,
reduce side effects. As disclosed herein (see also Background
Section and Examples), the glycosylceramide adjuvant of the
invention is not only highly immunostimulating at relatively low
doses (e.g., 10-100 .mu.g of the adjuvant per kg of the body
weight) but also possesses low toxicity and does not produce
significant side effects.
[0149] As specified above, the data obtained from the animal
studies can be used in formulating a range of dosage for use in
humans. The therapeutically effective dosage of
glycosylceramide-containing compositions of the present invention
in humans lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage can vary within this range depending upon the
dosage form employed and the route of administration utilized.
Ideally, a single dose should be used.
EXAMPLES
[0150] The following Example illustrates the invention without
limiting its scope.
Example 1
The Natural Killer T Cell Ligand .alpha.-Galactosylceramide
Enhances and Prolongs the Duration of Protective Immunity Induced
by Malaria Vaccines
[0151] Methods
[0152] Parasites and Their Use for Immunization and Challenge.
[0153] P. yoelii (17X NL strain) sporozoites were obtained by
dissecting the mosquito salivary glands as described (Rodrigues et
al., Int. Immunol., 3: 579-585, 1991; Gonzalez-Aseguinolaza et al.,
Proc. Natl. Acad. Sci. USA, 97: 8461-8466, 2000). For immunization,
sporozoites were radiation-attenuated by exposing them to 12,000
rad, and then injected intravenously into the tail vein or
subcutaneously into the base of the tail of the mice.
1.times.10.sup.4 and 1.times.10.sup.5 .gamma.-spz were used to
immunize mice for protection assay and an ELISPOT assay,
respectively. Parasitemia was determined by microscopic examination
of Giemsa stained thin blood smears obtained daily from day 3 to
day 14 post-sporozoite inoculation. Complete protection was defined
as the absence of parasitemia during this entire period.
[0154] Immunization with Recombinant Viruses.
[0155] A sub-optimal dose (1.times.10.sup.7 p.f.u.) of recombinant
adenovirus expressing the entire P. yoelii CS protein, AdPyCS
(Rodrigues et al., J. Immunol., 158: 1268-1274, 1997), was used to
immunize mice. The recombinant Sindbis virus expressing the CD8+T
cell epitope (SYVPSAEQI [SEQ ID NO: 2]) of P. yoelii CS protein,
SIN(Mal), and the recombinant Sinbis virus expressing the CD8+T
cell epitope (RGPGRAFVTI [SEQ ID NO: 5]) of HIV p18 protein,
SIN(P18), were constructed as described (Tsuji et al., J. Virol.,
72: 6907-6910, 1998; Villacres et al., Virology, 270: 54-64, 2000),
and 1.times.10.sup.5 p.f.u. of the viruses were inoculated s.c., as
a sub-optimal dose.
[0156] Mice.
[0157] BALB/c and B10.D2 mice were purchased from The Jackson
Laboratory (Bar Harbor, Me.) and maintained under standard
conditions in the Animal Facility. V.alpha.14 NKT-deficient mice
(J.alpha.281.sup.-/-) were established by specific deletion of the
J.alpha.281 gene segment with homologous recombination and
aggregation chimera techniques (Cui et al., Science, 278:
1623-1626, 1997; Gonzalez-Aseguinolaza et al., Proc. Natl. Acad.
Sci. USA, 97: 8461-8466, 2000) and used after 3-4 backcrosses to
BALB/c mice. CD1d-deficient mice (CD1d.sup.-/-) were generated from
embryonic stem cells of 129 origin and used after 7-8 backcrosses
to BALB/c (Mendiratta et al., Immunity, 6: 469-477,1997;
Gonzalez-Aseguinolaza et al., Proc. Natl. Acad. Sci. USA, 97:
8461-8466,2000). IFN-.gamma. receptor-deficient mice (IFN.gamma.
R.sup.-/-) were obtained from Swiss Institute for Experimental
Cancer Research (Epalinges, Switzerland), and used after 3
backcrosses to B10.D2 (Rodrigues et al., Parasite Immunol., 22:
157-160, 2000). Mice of either sex were used at 6-8 weeks.
[0158] .alpha.-GalCer.
[0159] .alpha.-GalCer,
[(2S,3S,4R)-1-O-(.alpha.-D-galactopyranosyl)-2-(N-h-
exacosanoylamino)-1,3,4-octadecanetriol], was synthesized by Kirin
Brewery (Gunma, Japan) using the method disclosed (Morita et al.,
J. Med. Chem., 38:2176-2187, 1995; Teriyuki et al., J. Med. Chem.,
42:1836-1841, 1999). The original product was dissolved in 0.5%
polysorbate-20 (Nikko Chemical, Tokyo) in 0.9% NaCl solution and
diluted with PBS just before use.
[0160] Peptide Immunization and .alpha.-GalCer Treatment.
[0161] Mice were immunized with peptides corresponding to the CD4+
T cell epitope (YNRNIVNRLLGDALNGKPEEK [SEQ ID NO: 1]) (Renia et
al., J. Immunol., 22: 157-160, 1993) or the CD8+ T cell epitope
(SYVPSAEQI [SEQ ID NO: 2]) (Rodrigues et al., Int. Immunol., 3:
579-585, 1991) of the P. yoelii CS protein. The peptide
representing the CD8+ epitope of the CS protein was emulsified in
incomplete Freund's adjuvant (IFA), while the peptide containing
the CS-specific CD4+ epitope was emulsified in complete Freund's
adjuvant (CFA). Mice were subcutaneously immunized with 10 mg of
the peptide. Some of the immunized mice were injected
intraperitoneally with .alpha.-GalCer, and others with vehicle
alone as a control. The dose and time of administration are
indicated below.
[0162] Quantification of Epitope-Specific CD4+ and CD8+ T Cells by
an ELISPOT Assay.
[0163] An ELISPOT assay was performed to determine the number of
CS-specific CD4+ and CD8+ T cells, producing IFN-.gamma. or IL-4
(Miyahira et al., J. Immunol. Methods, 181: 45-54, 1995). Briefly,
96 well nitrocellulose plates (Millipore, Bedford, Mass.) were
coated with anti-mouse IFN-.gamma. monoclonal antibodies (mAb), or
anti-mouse IL-4 mAb. After overnight incubation at room
temperature, the wells were washed repeatedly and blocked with
culture medium for 1 hour at 37.degree. C. The MHC-compatible
target cells, A20.2J B cell lymphoma, expressing both MHC class I
and II H-2.sup.d molecules, were incubated for 1 hour at 37.degree.
C. with the synthetic peptide representing the CD4+ T cell epitope
(YNRNIVNRLLGDALNGKPEEK [SEQ ID NO: 1]) or CD8+ T cell epitope
(SYVPSAEQI [SEQ ID NO: 2]) of the P. yoelii CS protein, or CD8+ T
cell epitope (RGPGRAFVTI [SEQ ID NO: 5]) of the HIV p18 protein.
After irradiating the peptide-pulsed target cells, the cells were
added to the ELISPOT wells. Untreated target cells were used as
negative controls. Serially diluted lymphocytes isolated from the
spleen or lymph nodes of immunized mice were co-cultured with
1.5.times.10.sup.5 target cells in the ELISPOT wells. After
incubating the plates 24 h for IFN-.gamma. detection or 48 h for
IL-4 detection at 37.degree. C. and 5% CO.sub.2, the plates were
treated as previously described (Rodrigues et al., J. Immunol.,
158: 1268-1274, 1997), and the number of spots corresponding to
IFN-.gamma. and IL-4 secreting cells determined.
[0164] Quantification of P. yoelii rRNA in the Liver of
Sporozoite-Inoculated Mice by Real-Time PCR.
[0165] Quantification of P. yoelii rRNA was performed as described
(Bruna-Romero et al., Int. J. Parasitol. 31: 1499-1502, 2001).
Briefly, total RNA was isolated by the method of Chomczynski and
Sacchi (Chomczynski and Sacchi, Anal. Biochem., 162: 156-159, 1987)
from the liver of mice sacrificed 42 hours after injection with
1.times.10.sup.4 P. yoelii sporozoites. After reverse transcription
of the extracted RNA, cDNA was generated and its amount analyzed by
real-time PCR, using the ABI Prism 5700 Sequence Detection
system(PE Biosystems, Foster City, Calif.; Bruna-Romero et al.,
Int. J. Parasitol., 31: 1499-1502, 2001). Primers and fluorogenic
probe with the following sequences were custom designed using the
ABI Prism primer Express software (PE Biosystems, Foster City,
Calif.), using P. yoelii (17XNL) 18S rRNA sequence (Bruna-Romero et
al., Int. J. Parasitol., 31: 1499-1502, 2001). The primers,
5'-GGGGATTGGT TTTGACGTTTTTGCG-3' (forward primer; SEQ ID NO: 17),
and 5'-AAGCATTAAATAAAG CGAATACATCCTTAT-3' (reverse primer; SEQ ID
NO: 18), were obtained from Operon Technologies Inc. (Alameda,
Calif.). The specific fluorogenic probe, PyNYU, 5'-FAM-CAATTG
GTTTACCTTTTGCTCTTT-TAMRA-3' (SEQ ID NO: 19), was obtained from PE
Applied biosystems (Foster City, Calif.), and was generated with
5-propyne-2'-deoxyuridine (turbo Taqman probe) to achieve a proper
Tm. The reaction mix contained 5 .mu.l of 10.times. Taqman buffer A
(PE Biosystems, Foster City, Calif.), 3.5 mM MgCl.sub.2, 200 .mu.M
dNTP, 0.3 .mu.M forward primer, 0.3 .mu.M reverse primer, 50 nM
turbo Taqman probe PyNYU, 1.25 U AmpliTaq Gold DNA polymerase, and
water up to 50 .mu.l final reaction volume. The temperature profile
included 95.degree. C. for 10 minutes and 35 cycles of denaturation
at 95.degree. C. for 15 seconds and annealing/extension at
60.degree. C. for 1 minute. The PCR products were visualized in 2%
agarose-1.times.TAE (50 mM Tris-acetate, pH 8.0, 1 mM EDTA) gels
stained with 0.5 mg/ml ethidium bromide. Digital images from the
gels were obtained using the Gel Doc 2000 gel documentation system
(BioRad, Hercules, Calif.), and analyzed by densitometry using
Quantity One software (BioRad, Hercules, Calif.). The precise
amount of parasite-derived 18S cDNA molecules detected in this
assay was determined by linear regression analysis using CT values
obtained from both liver samples and those obtained from a standard
curve generated with known amounts of plasmid 18S cDNA.
[0166] Quantification of .alpha.-GalCer-Specific Cells by ELISPOT
Assay.
[0167] The relative numbers of IFN-.gamma. and/or IL-4 producing
.alpha.-GalCer-specific lymphocytes were determined using an
ELISPOT assay. Lymphocytes were isolated from the liver of
wild-type and IFN-.gamma. R-deficient mice, as described (Rodrigues
et al., J. Immunol., 158: 1268-1274, 1997). After 12 hour
incubation with 100 ng/ml of .alpha.-GalCer or vehicle at a cell
density of 10.sup.7 cells/ml, serially diluted lymphocytes,
starting at 1.times.10.sup.6 cells per well, were placed into
ELISPOT wells coated with corresponding anti-cytokine antibodies.
After incubating the plates for 24 hours at 37.degree. C. and 5%
CO.sub.2, the plates were developed as described (Rodrigues et al.,
J. Immunol., 158: 1268-1274, 1997).
[0168] Flow Cytometric Analysis Using CD1d/.alpha.-GalCer
Tetramers.
[0169] .alpha.-GalCer-specific lymphocytes were identified using
CD1d/.alpha.-GalCer tetrameric complexes, consisting of CD1d
molecules and .alpha.-GalCer, as previously described (Matsuda et
al., J. Exp. Med., 192: 741-754, 2000). Freshly isolated hepatic
lymphocytes were incubated first with phycoerythrin (PE)-labelled
tetrameric complexes, followed by a second incubation with
FITC-labelled anti-CD3 monoclonal antibody. The cells were then
analyzed by a FACScalibur instrument (Becton Dickinson, San Jose,
Calif.) using CELLQUEST software (Becton Dickinson).
[0170] Indirect Immunoflurescence Assay (IFA).
[0171] Sera of immunized mice were obtained just before their
challenge with sporozoites, and their Ab titers were measured using
P. yoelii sporozoites in an indirect immunofluorescence assay
(IFA). In brief, P. yoelii sporozoites were placed on multispot
glass slides, and air-dried. After 1 hour of incubation with the
sera, diluted in PBS containing 1% BSA, the slides were washed with
PBS and incubated 1 hour with FITC- labelled affinity purified goat
anti-mouse Ab (Kirkegaard & Perry Laboratories, Gaithersburg,
Md.). The slides were then washed and mounted in PBS containing 50%
(v/v) glycerol and 1% (w/v) Evans blue to reduce bleaching. The
highest serum dilution resulting in fluorescence of the sporozoites
was considered to be the IFA titer.
[0172] Measurement of Ab Isotype Level by ELISA.
[0173] Sera of immunized mice were obtained prior to their
challenge with sporozoites, and the levels of CS-specific IgM,
IgG1, IgG2a, and IgE isotypes were measured using the Mouse
Hybridoma Subtyping Kit (Boehringer Mannheim, Mannheim, Germany).
Briefly, plates were coated with 10 mg/ml of B cell epitope
(QGPGAP).sub.2 (Charoenvit et al., J. Immunol., 146: 1020-1025,
1991) of the P. yoelii CS protein, blocked with PBS containing 1%
BSA, and incubated for 1 hour with 1:5 dilution of sera from
immunized and non-immunized mice. The plates were then washed and
anti-mouse IgM, IgG1a, IgG2a (Boehringer Mannheim) and IgE
(Southern Biotechnology Associates, Inc., Birmingham, Ala.)
conjugated to peroxidase were added, followed by incubation with
the substratre 2,2-Azino-di-[3-ethylbenzthiazoline sulfonate (6)],
according the manufacture's instruction.
[0174] Statistical Analysis.
[0175] Student's t test was used for all comparisons. Only P values
below 0.01 were considered significant. Data are presented as mean
values.+-.SD.
Results
.alpha.-GalCer Enhances Protective Anti-Malaria Immunity
[0176] To assess the ability of .alpha.-GalCer to enhance the
protective anti-malaria immune response induced by immunization
with a suboptimal dose of irradiated sporozoites, BALB/c mice were
immunized intravenously with a sub-optimal dose (1.times.10.sup.4)
of irradiated sporozoites (.gamma.-spz) together with different
doses of .gamma.-GalCer (0.5, 1, 2 .mu.g). Two weeks later, these
different groups of mice were challenged with 1.times.10.sup.4 live
P. yoelii sporozoites, and the levels of protective anti-malaria
immunity were measured by determining the amount of
parasite-specific rRNA in the liver using a highly sensitive real-
time PCR assay (Bruna Romero et al., Int. J. Parasitol. 31: 1499
1502, 2001). .alpha.-GalCer administration significantly enhanced,
in a dose-dependent manner, the level of protective immunity (%
inhibition of the liver stage development) elicited by immunization
with .gamma.-spz (FIG. 1A). Indeed, the parasite load in the livers
of .gamma.-spz-immunized mice administered with 2 .mu.g of
.alpha.-GalCer was 10 times smaller than that in the livers of mice
immunized with .gamma.-spz alone.
[0177] The present inventors also determined the titers of
anti-sporozoite antibodies using an immunofluorescence assay(IFA)
of air-dried P. yoelii sporozoites, as well as the titers of
antibody against the circumsporozoite (CS) protein, the major
surface antigen of sporozoites, using ELISA. The antibody titers
were identical among the groups of .gamma.-spz-immunized mice
regardless of whether or not they received .alpha.-GalCer (FIG.
1A). When the immunoglobulin isotype of the anti-CS antibodies was
determined, no significant differences in IgE, IgG.sub.1,
IgG.sub.2a or IgM isotype profiles of anti-CS antibodies were
detected between .alpha.-GalCer-treated and untreated mice. These
results indicate that anti-malarial humoral response is not
affected by .alpha.-GalCer treatment.
[0178] The kinetics of the adjuvant activity displayed by
.alpha.-GalCer were then examined by administering 2 .mu.g of the
glycolipid to BALB/c mice on the same day, two days before or two
days after intravenous immunization with 1.times.10.sup.4
.gamma.-spz. The highest level of protective anti-malaria immunity
was elicited when .alpha.-GalCer was administered on the same day
as .gamma.-spz (FIG. 1B). The administration of .alpha.-GalCer two
days after .gamma.-spz immunization did not significantly enhance
the level of protective immunity induced by sporozoites alone.
Interestingly, when .alpha.-GalCer was administered two days prior
to .gamma.-spz immunization, protective immunity was completely
abolished. It is possible that .alpha.-GalCer administered two days
earlier might have eliminated the sporozoites before they could be
processed and presented by antigen presenting cells, thereby
preventing the induction of a malaria-specific immune response. As
shown in the previous study by the present inventors
(Gonzalez-Aseguinolaza et al., Proc. Natl. Acad. Sci. USA, 97:
8461-8466, 2000), .alpha.-GalCer administered two days prior to
challenge with live sporozoites completely eliminates the parasites
from the liver in a manner dependent on NKT cells and IFN-.gamma..
Similar kinetics of the adjuvant activity of .alpha.-GalCer were
observed in B10.D2 mice. (FIG. 1B).
Co-Administration of .alpha.-GalCer with a Malaria Antigen Enhances
Malaria-Specific T Cell Responses, Particulary Those of CD8+T
Cells
[0179] To determine whether .alpha.-GalCer-mediated enhancement of
the protective immune response against malaria is a particular
phenomenon related to .gamma.-spz immunization, or a more general
phenomenon independent of the immunogen administered,
.alpha.-GalCer was administered to BALB/c mice on the same day as
subcutaneous immunization with a sub-optimal dose of recombinant
adenovirus expressing the whole CS protein, AdPyCS (Rodrigues et
al., J. Immunol., 158: 1268-1274,1997), or recombinant Sindbis
virus expressing the CD8+ T cell epitope of the CS protein,
SIN(Mal) (Tsuji et al., J. Virol. 72: 6907-6910, 1998). As shown in
FIGS. 1C and 1D, .alpha.-GalCer significantly enhances the
protective immune response induced by immunization with a
sub-optimal dose of the two different recombinant viruses. In the
case of AdPyCS, the protection was augmented almost 10 times to
that of control, and in the case of SIN(Mal), the protection after
co-administration with .alpha.-GalCer was enhanced 3 times.
[0180] To further assess the adjuvant activity of .alpha.-GalCer
co-administered with vaccines, parasitemia (i.e., the presence of
parasites in the blood) was monitored daily by microscopic
examination of thin blood smears. Briefly, BALB/c mice were
immunized either intravenously with 1.times.10.sup.4 .gamma.-spz or
subcutaneously with 1.times.10.sup.7 p.f.u. of AdPyCS, doses which
otherwise fail to confer protection against malaria, with or
without .alpha.-GalCer treatment. Two weeks later, all mice were
challenged with 50 viable P. yoelii sporozoites, and determined the
occurrence of blood infection by monitoring parasitemia. 28 out of
30 .alpha.-GalCer-treated, .gamma.-spz-immunized mice were
protected, while most of the .alpha.-GalCer-untreated,
.gamma.-spz-immunized mice developed malaria infection (Table I).
Similarly, administration of .alpha.-GalCer together with AdPyCS
strongly enhanced the protective effect induced by a sub-optimal
dose of the virus. On the other hand, administration of
.alpha.-GalCer alone failed to protect the challenged mice.
Overall, these results corroborate the liver stage data (FIG. 1),
and together indicate that .alpha.-GalCer administration increases
the efficacy of a sub-optimal immunizing dose of both .gamma.-spz
and recombinant viruses, revealing a profound adjuvant effect.
1TABLE I .alpha.-GalCer Enhances Protective Immunity Induced by
Malaria Immunogens Number of mice protected/ % protection Immunogen
number challenged (no parasitemia) .gamma.-spz* 6/30 20 .gamma.-spz
+ .alpha.-GalCer 28/30 93 AdPyCS* 2/30 7 AdPyCS + .alpha.-GalCer
24/30 80 .alpha.-GalCer 0/30 0 None 0/30 0 *BALB/c mice were
immunized either intravenously with 1 .times. 10.sup.4 .gamma.-spz
or subcutaneously with 1 .times. 10.sup.7 p.f.u. of AdPyCS.
.alpha.-GalCer Enhances T Cell Responses Elicited by Various
Vaccines
[0181] In order to determine which components of the
malaria-specific T cell responses (i.e., CD4+ and/or CD8+ T cell
responses) are enhanced by co-injection of .alpha.-GalCer with
.gamma.-spz, these immune parameters were compared in
.gamma.-spz-immunized mice treated with or without .alpha.-GalCer.
For this purpose, BALB/c mice were immunized with 1.times.10.sup.5
.gamma.-spz, either together with a vehicle (0.5% polysorbate-20,
Nikko Chemical, Tokyo) or .alpha.-GalCer. Two or six weeks later,
splenic lymphocytes were isolated, and the numbers of CS-specific,
IFN-.gamma.- and IL-4-secreting CD8+ and CD4+T cells were
determined by an ELISPOT assay (Rodrigues et al., J. Immunol., 158:
1268-1274, 1997). As shown in FIG. 2A, .alpha.-GalCer treatment
strikingly enhanced the level of CS-specific T cell responses
elicited by .gamma.-spz at two weeks after immunization.
Specifically, .alpha.-GalCer increased the number of
IFN-.gamma.-secreting CS-specific CD8+ T cells approximately seven
fold compared to those induced by .gamma.-spz immunization alone
(FIG. 2A). Furthermore, the number of IFN-.gamma.-secreting
CS-specific CD4+ T cells was also significantly increased, albeit
to a lesser degree (FIG. 2A). More importantly, the administration
of .alpha.-GalCer not only enhanced the level of CS-specific CD8+ T
cell response but also prolonged the duration of the response (FIG.
2A; see below). Such strong enhancement of the T cell responses by
.alpha.-GalCer treatment was not observed when .alpha.-GalCer was
administered two days prior to or two days after the .gamma.-spz
immunization. No difference was found in the numbers of CS-specific
CD4+ or CD8+ T cells secreting IL-4, indicating that .alpha.-GalCer
treatment primarily enhances antigen-specific Th1-type responses in
the present experimental system. Because similar results were
obtained in both BALB/c and B10.D2 mice, it can be concluded that
the adjuvant effect of .alpha.-GalCer is not influenced by the
different genetic backgrounds of these mice.
[0182] Since it was found that .alpha.-GalCer administration
strongly augments the level of CS-specific T cell responses in
sporozoite-immunized mice, the present inventors have decided to
determine whether .alpha.-GalCer could also enhance CS-specific T
cell responses upon peptide immunization as well as upon
immunization with recombinant viruses. .alpha.-GalCer was
administered to BALB/c mice at the same time as subcutaneous
immunization with (i) 10 mg of a synthetic peptide corresponding to
either the CD8+ T cell epitope or the CD4+ T cell epitope of the CS
protein or (ii) suboptimal dose of AdPyCS. Ten days later, lymph
node cells (for peptide immunization) and splenocytes (for viral
immunization) were obtained from these groups of mice, and the
numbers of CS-specific T cells secreting IFN-.gamma. or IL-4 were
determined by an ELISPOT assay. The number of both CS-specific CD4+
and CD8+ T cells secreting IFN-.gamma. elicited in
.alpha.-GalCer-treated, peptide-immunized mice was significantly
higher than the number of such T cells in peptide-immunized mice
without .alpha.-GalCer treatment. .alpha.-GalCer administered two
days before or two days after the peptide immunization was also
able to significantly enhance the CS-specific T cell responses,
albeit to a lesser degree than the responses enhanced by
simultaneous administration of .alpha.-GalCer with the peptides.
The number of both CS-specific CD4+ and CD8+ T cells secreting
IFN-.gamma. elicited in .alpha.-GalCer-treated AdPyCS-immunized
mice was more than 10-fold higher than that of T cells from a group
of mice immunized with the virus alone (FIG. 2B). When SIN(Mal) was
used, it was found that .alpha.-GalCer treatment also increases the
number of CS-specific CD8+ T cells secreting IFN-.gamma. (FIG.
2C).
[0183] The present inventors have also examined whether the
adjuvant activity of .alpha.-GalCer is a phenomenon related
specifically to the H-2Kd-restricted CD8+ T cell epitope of the CS,
or can be applied to non-malarial epitopes. For this purpose BALB/c
mice were immunized with a recombinant Sindbis virus expressing a
H-2Dd-restricted CD8+ T cell epitope (RGPGRAFVTI [SEQ ID NO: 5]) of
p18 protein (V3 loop) of HIV (Villacres et al., Virology, 270:
54-64, 2000). .alpha.-GalCer co-administration increased the number
of p18-specific IFN-.gamma.-secreting CD8+ T cells induced by
immunization with SIN(p18) 4-fold (FIG. 2C). These results indicate
that (i) .alpha.-GalCer treatment enhances a CD8+ T cell response
specific for HIV antigen in mice, and that (2) .alpha.-GalCer
treatment enhances a CD8+ T cell response induced by a recombinant
Sindbis virus expressing a foreign epitope, i. e., another form of
antigen presentation. More generally, the data presented herein
demonstrate that the enhancement of the cellular immune response by
treatment with .alpha.-GalCer is independent of the antigen
delivery system (attenuated pathogen, peptide or recombinant virus)
and the epitope.
.alpha.-GalCer Prolongs the Duration of Both Malaria-Specific T
Cell Responses and Anti-Malaria Protection Elicited by Sporozoite
Immunization
[0184] Next, the duration of the CS-specific CD8+ T cell responses
was compared in .alpha.-GalCer-treated, sporozoite-immunized mice,
and in sporozoite-immunized mice without .alpha.-GalCer treatment.
BALB/c mice were immunized with 1.times.10.sup.5 .gamma.-spz, with
or without .alpha.-GalCer-treatment, and two or four weeks later,
splenocytes were obtained from these mice and the number of
CS-specific CD8+ T cells secreting IFN-.gamma. determined by an
ELISPOT assay. The administration of .alpha.-GalCer not only
enhanced the level of the CS-specific CD8+ T cell response, but
also prolonged the duration of this response (FIG. 3A).
[0185] To determine the adjuvant effect of .alpha.-GalCer on the
duration of protection against malaria, parasitemia (i.e., the
presence of the parasites in the blood) was monitored daily via
microscopic inspection of thin blood smears in two separate
experiments. In experiment 1, two groups of mice, one treated with
.alpha.-GalCer and the other untreated, were immunized with
1.times.10.sup.4 .gamma.-spz, a dose that fails to confer
protection against malaria 2 weeks after immunization. Two weeks
later, all mice were challenged with 50 viable P. yoelii
sporozoites, and the occurrence of blood infection was determined
by monitoring parasitemia. In experiment 2, two groups of mice, one
treated with a-GalCer and the other untreated, were immunized with
1.times.10.sup.5 .gamma.-spz, a dose that induces complete
protection 2 weeks after immunization but not 4 weeks after. Four
weeks later, these immunized mice, as well as naive controls were
challenged with 50 live sporozoites, and the course of infection
was determined as described above. It was found that nine out of
ten .alpha.-GalCer-treated, sporozoite-immunized mice were
protected in both experiments, while most of the
sporozoite-immunized mice that did not receive
.alpha.-GalCer-treatment developed malaria infection (FIG. 3B).
These results corroborate data obtained in studies determining the
parasite burden in the liver by RT-PCR, and further demonstrate
that .alpha.-GalCer administration prolongs the duration of a
protective immune response and increases the efficacy of a
sub-optimal immunizing dose of irradiated sporozoites, revealing an
adjuvant effect.
The Activity of .alpha.-GalCer Requires CD1D Molecules, V.alpha.14
NKT Cells and IFN-.gamma.
[0186] As .alpha.-GalCer has been shown to activate NKT cells in
the context of CD1d molecules (see the references from the
Background Section, e.g., Kawano et al., Science, 278: 1626-1629,
1997), cellular mechanism underlying .alpha.-GalCer's adjuvant
activity was investigated using mice lacking CD1d molecules as well
as mice deficient in T cells expressing the canonical NKT cell
receptor. Briefly, these knockout mice, along with wild-type
controls, were immunized with a suboptimal dose of .gamma.-spz
(1.times.10.sup.4) together with or without .alpha.-GalCer
treatment. Two weeks later, these immunized mice, as well as
non-immunized controls were challenged with live sporozoites, and
the adjuvant levels of protective anti-malaria immunity were
determined. As shown in FIG. 4A, the administration of
.alpha.-GalCer, which increased the level of .gamma.-spz-induced
protective immunity in wild-type mice, failed to enhance the
protective immunity in CD1d-deficient mice, as well as in
J.alpha.281-deficient mice, which lack V.alpha.14 NKT cells. These
results indicate that the adjuvant activity of .alpha.-GalCer is
dependent on both CD1d molecules and V.alpha.14 NKT cells.
[0187] To further demonstrate the importance of CD1d molecules and
V.alpha.14 NKT cells for the adjuvant activity of .alpha.-GalCer,
the number of CS-specific CD8+ T cells was measured in
.gamma.-spz-immunized, .alpha.-GalCer-treated or untreated mice,
deficient in either CD1d or V.alpha.14 NKT cells. As shown in FIG.
4B, .alpha.-GalCer treatment failed to increase the number of
CS-specific CD8+ T cells induced by .gamma.-spz immunization in
CD1d-deficient mice compared to that of untreated mice, indicating
that .alpha.-GalCer requires CD1d to enhance the CS-specific CD8+ T
cell response. Interestingly, in .gamma.-spz-immunized and
.alpha.-GalCer-treated, J.alpha.281-deficient mice, the number of
CS-specific CD8+ T cells was significantly increased compared to
that of untreated mice (FIG. 4B). However, this increase did not
reach the level of .alpha.-GalCer-treated, .gamma.-spz-immunized
wild-type mice (FIG. 4B) and did not enhance the level of
protective anti-malaria immunity (FIG. 4A). These findings,
therefore, demonstrate the importance of V.alpha.14 NKT cells in
mediating the adjuvant effect of .alpha.-GalCer.
[0188] Lastly, in order to gain insight into the molecular
mechanism underlying .alpha.-GalCer's adjuvant activity, mice
lacking the IFN-.gamma. receptor (IFN-.gamma. R.sup.-/-) were
immunized with .gamma.-spz with or without .alpha.-GalCer
co-treatment, and ten days later, the numbers of CS-specific
IFN-.gamma.-secreting CD8+ and CD4+ T cells were analyzed using an
ELISPOT assay. .alpha.-GalCer co-administration failed to augment
the number of CS-specific IFN-.gamma.-secreting CD8+ and CD4+ T
cells in the .gamma.-spz-immunized knockout mice (FIG. 5A). It has
been reported that mice deficient in different molecules such as
GM-CSF receptor .beta.-chain (Sato et al., Proc. Natl. Acad. Sci.
USA, 96: 7439-7444, 1999) and Fas (Mieza et al., J. Immunol., 156:
4035-4040, 1996) are also partially deficient in NKT cells. To
exclude the possibility that the absence of the IFN-.gamma.
receptor results in a decreased number and/or defective function of
NKT cells, the presence and the function of NKT cells in these
IFN-.gamma.R.sup.-/- mice was analyzed by CD1d/.alpha.-GalCer
tetramer staining and ELISPOT assay. Flow cytometric analysis using
CD1d/.alpha.-GalCer tetramers revealed that the percentage of
.alpha.-GalCer-specific NKT cells among hepatic lymphocytes in
IFN-.gamma.R.sup.-/- mice is similar to that in wild-type mice
(FIG. 5B). In addition, the number of .alpha.-GalCer specific cells
secreting IFN-.gamma.in the liver (FIG. 5C) and spleen of wild-type
and IFN-.gamma.R.sup.-/-mice is similar, eliminating the
possibility that the lack of adjuvant activity was due to a defect
in the NKT cell population. Collectively, these results indicate
that .alpha.-GalCer's adjuvant activity is dependent on IFN-.gamma.
production.
Discussion
[0189] The present Example addresses the ability of the NKT cell
ligand, .alpha.-GalCer, to act as an adjuvant to modulate acquired
anti-malaria immunity induced by malaria-specific antigen(s). As
disclosed herein, .alpha.-GalCer administration to mice immunized
with sub-optimal doses of (i) irradiated Plasmodium yoelii
sporozoites, (ii) synthetic peptides corresponding to either the
CD8+ T cell epitope or the CD4+ T cell epitopes of the CS protein
or (iii) recombinant viruses expressing the whole CS protein or the
CD8+ T cell epitope of the CS protein greatly enhances protective
anti-malaria immunity. In addition, .alpha.-GalCer-treatment was
found herein to elicit a higher level of protection even four weeks
after sporozoite immunization, indicating that a longer lasting
protective immunity could be elicited by the conjoint
administration of .alpha.-GalCer.
[0190] The main immune components affected by the .alpha.-GalCer
administration appear to be malaria-specific CD8+ and CD4+ T cells
that secrete IFN-.gamma.. In the present study, the levels of the
humoral response as well as the Th2-type response were unaltered by
the .alpha.-GalCer treatment. In contrast, the administration of
.alpha.-GalCer increased the number of IFN-.gamma.-secreting
CS-specific CD4+ and CD8+ T cells induced by .gamma.-spz
immunization approximately 5-fold and 7-fold, respectively.
Furthermore, the level of the CS-specific T cell responses remained
much higher at six weeks after .gamma.-spz immunization in
.alpha.-GalCer-treated mice compared to that in non-treated mice.
Since protective immunity against the liver stages of malaria is
primarily mediated by CD8+ T cells as well as CD4+ T cells, and
requires production of IFN-.gamma. (Schofield et al., Nature 330:
664-666, 1987; Weiss et al., Proc. Natl. Acad. Sci. USA, 85:
573-576,1988; Doolan and Hoffman. J. Immunol., 165: 1453-1462,
2000), it is not surprising that the level of anti-malaria
protection is increased and its duration prolonged by the
.alpha.-GalCer treatment.
[0191] The adjuvant effect of .alpha.-GalCer was also observed when
the .alpha.-GalCer-treated mice were immunized with synthetic
peptides corresponding to the CD4+ and CD8+ epitopes or with
recombinant viruses expressing either the P. yoelii CS protein or
the H-2Kd-restricted CD8+ T cell epitope of this protein. These
results confirm and extend the data obtained by .gamma.-spz
immunization, indicating that malaria-specific CD8+ and CD4+ T cell
responses are enhanced by .alpha.-GalCer administration, regardless
of the type of immunogen used (whole parasite, or peptide, or
recombinant virus). It is also demonstrated herein that the CD8+ T
cell response enhanced by .alpha.-GalCer administration is
independent of the CD8+ T cell epitope used, since the immune
response induced by a recombinant Sindbis virus expressing a
H-2Dd-restricted T cell epitope of HIV was also enhanced.
[0192] .alpha.-GalCer's ability to augment the level of protective
anti-malaria immunity induced by .gamma.-spz immunization requires
both CD1d molecules and V.alpha.14 NKT cells. Without these
components, .alpha.-GalCer was unable to increase the protection
elicited by a sub-optimal dose of the immunogen. Although both CD1d
molecules and V.alpha.14 NKT cells were needed for .alpha.-GalCer's
ability to augment protective anti-malaria immunity, a noticeable
increase in the number of CS-specific CD8+ T cells was detected in
J.alpha.281-deficient mice after .alpha.-GalCer-treatment. This may
be due to the high degree of genetic heterogeneity of these mice,
which affects the T cell response and causes this moderate
increase. Alternatively, CD1d-reactive, non-V.alpha.14 NKT cells
may exist in J.alpha.281-deficient mice.
[0193] While the precise molecular mechanism of the adjuvant effect
of .alpha.-GalCer remains to be fully clarified, the present
finding that these activities of .alpha.-GalCer are eliminated in
mice lacking IFN-.gamma. receptor indicates that IFN-.gamma. is
important in mediating the adjuvant effect of .alpha.-GalCer. It is
possible that IFN-.gamma. secreted by NKT and/or NK cells acts on
antigen presenting cells, by up-regulating the MHC class I
processing machinery, e.g., TAP, proteasome subunits and class I
heavy chains. Alternatively, IFN-.gamma. may enhance the acquired
cell-mediated immune response by directly acting on
antigen-specific CD8+ T cells.
[0194] The instant kinetic studies demonstrate that .alpha.-GalCer
displays a maximal adjuvant effect only when the glycolipid is
co-administered with an antigen (such as irradiated plasmodial
sporozoites [.gamma.-spz], or malaria-specific peptide epitopes
[e.g., presented by recombinant viruses]) and that the
administration of .alpha.-GalCer two days prior to or two days
after the immunization with these immunogens led to lack of
adjuvant activity. A recent study on the in vivo kinetics of NKT
cells after .alpha.-GalCer administration using CD1d/.alpha.-GalCer
tetramers, has indicated that murine NKT cells, especially those in
the liver where they constitute 20-30% of the lymphocyte
population, are promptly activated, secrete large amounts of
IFN-.gamma. and IL-4, and readily disappear 5 hours after the
stimulation (Matsuda et al., J. Exp. Med., 192: 741-754, 2000).
Interestingly, this acute disappearance of .alpha.-GalCer-activated
NKT cells was also confirmed by phenotypic analysis of the
peripheral blood of cancer patients treated with
.alpha.-GalCer.
[0195] As previously shown by various investigators, NKT cell
activation not only causes activation of NK cells but also
proliferation of memory CD4+ and CD8+ T cells (Eberl et al., J.
Immunol., 165: 4305-4311, 2000) or induction of the early
activation marker CD69 on the surface of T cells and B cells
(Nishimura et al., Int. Immunol. 12: 987-994, 2000), suggesting a
role for activated NKT cells in initiating T cell and B cell
responses. Also, recent studies by a number of different
investigators indicate that IFN-.gamma. is secreted by both NKT and
NK cells after .alpha.-GalCer treatment (Nishimura et al., Int.
Immunol. 12: 987-994, 2000; Camaud et al., J. Immunol., 163:
4647-4650, 1999; Eberl and MacDonald, Eur. J. Immunol., 30:
985-992, 2000). In one of these studies it has been shown that
administration of .alpha.-GalCer to mice immunized with a T cell
lymphoma enhances the generation of tumor-specific cytotoxic T
cells (Nishimura et al., Int. Immunol. 12: 987-994,2000). However,
as to whether .alpha.-GalCer-activated NKT cells can contribute to
the induction of protective immunity against pathogens or tumors
has not been elucidated. In this regard, the present study
indicates for the first time that .alpha.-GalCer-activated NKT
cells play a role in the induction of protective immunity, in which
specific CD8+ T cells are the primary effector cells.
[0196] In conclusion, this study has shown that .alpha.-GalCer acts
as an adjuvant to enhance and/or extend the duration of protective
antigen-specific immune responses. Specifically, as disclosed
herein, this .alpha.-GalCer-mediated immunostimulation is at least
in part attributed to the .alpha.-GalCer-activated NKT cells.
Although an endogenous mammalian counterpart of .alpha.-GalCer has
yet to be identified, it is conceivable that it would be also
induced under a range of pathological and inflammatory conditions
to activate NKT cells (Mieza et al., J. Immunol., 156:4035-4040,
1996; Sumida et al., J. Exp. Med., 182:1163-1168, 1995).
Accordingly, the studies disclosed herein may present evidence for
a role of NKT cells in bridging innate and adaptive immunity.
[0197] The present findings on the adjuvant activity of
.alpha.-GalCer are clearly applicable not only to malaria, but also
to various other intracellular microbial pathogens, as well as to
other infections and tumors. Finally, since it has been
demonstrated that .alpha.-GalCer can stimulate not only murine but
also human NKT cells (Brossay et al., J. Exp. Med. 188: 1521-1528,
1998; Spada et al., J. Exp. Med., 188: 1529-1534, 1998), instant
findings can be directly applied to the understanding of the role
of human NKT cells, and the design of novel, more effective human
vaccines.
[0198] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0199] All patents, applications, publications, test methods,
literature, and other materials cited herein are hereby
incorporated by reference.
Sequence CWU 1
1
19 1 21 PRT P. yoelii 1 Tyr Asn Arg Asn Ile Val Asn Arg Leu Leu Gly
Asp Ala Leu Asn Gly 1 5 10 15 Lys Pro Glu Glu Lys 20 2 9 PRT P.
yoelii 2 Ser Tyr Val Pro Ser Ala Glu Gln Ile 1 5 3 8 PRT P.
falciparum 3 Asn Val Asp Pro Asn Ala Asn Pro 1 5 4 20 PRT P.
falciparum 4 Glu Tyr Leu Asn Lys Ile Gln Asn Ser Leu Ser Thr Glu
Trp Ser Pro 1 5 10 15 Cys Ser Val Thr 20 5 10 PRT HIV-1 5 Arg Gly
Pro Gly Arg Ala Phe Val Thr Ile 1 5 10 6 11 PRT HIV-1 6 Lys Ala Phe
Ser Pro Glu Val Ile Pro Met Phe 1 5 10 7 8 PRT HIV-1 7 Lys Ala Phe
Ser Pro Glu Val Ile 1 5 8 9 PRT HIV-1 8 Thr Pro Gln Asp Leu Asn Met
Met Leu 1 5 9 9 PRT HIV-1 9 Thr Pro Gln Asp Leu Asn Thr Met Leu 1 5
10 10 PRT HIV-1 10 Asp Thr Ile Asn Glu Glu Ala Ala Glu Trp 1 5 10
11 10 PRT HIV-1 11 Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys 1 5 10
12 9 PRT HIV-1 12 Gln Ala Thr Gln Glu Val Lys Asn Trp 1 5 13 9 PRT
HIV-1 13 Arg Leu Arg Pro Gly Gly Lys Lys Lys 1 5 14 9 PRT HIV-1 14
Ser Leu Tyr Asn Thr Val Ala Thr Leu 1 5 15 12 PRT P. falciparum 15
Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro 1 5 10 16 9 PRT
influenza A virus 16 Thr Tyr Gln Arg Thr Arg Ala Leu Val 1 5 17 25
DNA Artificial Sequence forward primer for RT-PCR 17 ggggattggt
tttgacgttt ttgcg 25 18 30 DNA Artificial Sequence reverse primer
for RT-PCR 18 aagcattaaa taaagcgaat acatccttat 30 19 24 DNA
Artificial Sequence fluorogenic probe, PyNYU 19 caattggttt
accttttgct cttt 24
* * * * *